Vampirism is spread by the ingestion of vampire blood or by its direct introduction to the human blood stream. These are currently the only possible methods of infection (tactile contact with Eve is no longer possible since her death), since many hunters are seen getting splashed in the face with vampire blood without being turned. This means that the vampire blood doesn't enter a person's system through the eyes, nose or unbroken skin. It may also mean that, in terms of ingestion, a certain quantity is required to cause the transformation, considering getting splattered blood on the lips may result in some entering the mouth, becoming mixed with saliva, and winding up swallowed. These limitations are very beneficial to hunters due to the fact that killing a vampire is usually a gory and messy procedure. According to Dean Winchester, "the change, sometimes its fast, sometimes its slow, it just depends on the person."

Newly turned vampires are always confused and disoriented, especially while adjusting to the overwhelmingly sharp senses and sensitivity to light. Bloodlust usually sets in after, and drives the newly turned vampire into a killing spree. They then normally seek the one who changed them, known as their maker, and usually accept them as their leader. Most of the vampires shown in the series develop or are integrated into a nest or hive mentality. These newly turned vampires are still half-human until they drink their first blood.

Vampirism is rarely curable, with the only known cure being a potion recorded in a journal by an ancestor of the Campbell Family of hunters. The ritual calls for the blood of the new vampire's sire, and will not work if the subject has fed on human blood. Other known ingredients include garlic and sage. The reversion is painful, but no secondary effects are retained. At least one cured vampire has retained all of his memories before he was reverted to humanity. However, the cure causes you to be sick for a few days. The cure does not require the vampire that turned the person to be alive for it to work.

This technique was used to cure Dean Winchester, Seth's Victim, Annie Jones and Doug Stover.

A vampire called Dixon has been infecting young blonde women with vampirism in the hopes of rebuilding his lost family and repopulating the vampiric species. He converts them by feeding them his blood under the pretense that it is a recreational drug, administered either with an eyedropper or in a drink. One of the girls he has recently turned is caught and interrogated by Sam and Dean, which puts them on his trail, but the timely arrival of a vengeful Gordon prevents them from pursuing Dixon.

In a cruel twist of fate, Dixon captures Gordon and turns him into a vampire as punishment for the vampires he's killed in the past. Gordon is not a passive victim, however, and he uses his new strength to murder Dixon's girls and to escape. This drives Dixon to despair and apathy, and when the Winchesters find his lair, he admits to everything he has done. It is implied that the hunters killed him. Gordon, after escaping Dixon's lair, soon falls victim to bloodlust and feeds on human blood. He then continues his hunt for Sam by setting a trap, capturing and infecting another woman to work as a distraction and then isolating Sam so that he can attack him. Gordon underestimates Sam, however, and is subsequently decapitated with barbed wire. His victim is killed by Dean with the Colt after a struggle.

In Limestone, Illinois, Sam and Dean investigate the disappearances of several young women. They discover that the most recent victim was obsessed with Twilight-style vampires, and that she was communicating with someone who claimed to be a vampire online. At the same time, a blood bank van is robbed. Sam and Dean go to a club called the Black Rose, the girl's last destination, and discover vampires trying to "recruit" new members. Sam kills one vampire, but lets Dean be turned by a vampire named Boris. The Winchesters return to their hotel room, and Sam calls Samuel. Dean, who is prepared to be killed by Samuel, sneaks away to bid Lisa goodbye. He is nearly overwhelmed by his hunger, but resists and returns to Sam and Samuel. Samuel surprises Dean by revealing a cure in one of his ancestor's hunting journals. In order to work, Dean must consume no human blood, and must acquire the blood of the vampire that turned him, so Dean goes to the nest of the vampires with a syringe of dead man's blood.

At the nest, a "recruiter" named Robert offers Dean blood, which he declines, and reveals that they are no longer allowed to kill people. Dean meets Boris, and tries to stab him with the dead man's blood, but Boris is too quick. Before Boris can kill Dean, all the vampires collapse, Dean included. He has a shared vision of the Alpha Vampire, and perceives that the Alpha wishes to build an army. When he awakes, Dean fights the vampires in the nest. He eventually kills all the members, except one beheaded by Sam and Samuel, and acquires Boris's blood. Samuel prepares the cure and Dean drinks it. He vomits blood and passes out, but re-awakens as a human.

Samuel Campbell and his people capture the Alpha Vampire. They subdue him using an I.V. of Dead Man's Blood and attempt to interrogate him, but he is extremely resistant to pain and doesn't give Samuel any information. After Samuel leaves the room where he is being held, he is approached by Dean and Sam and he reveals to them that, though he is the first vampire, he has a mother. He also tells them that Samuel is torturing him for information about Purgatory and its location. Shortly after their conversation, he frees himself from his cage and almost manages to flee the area, but demons appear and take him away on Crowley's orders. It is revealed that Samuel has been working for Crowley, who takes Alpha Vampire to continue interrogating it about Purgatory. Apparently he is unable to get anything from him as in "All Dogs Go To Heaven," he sends them after what he believes to be werewolves in hopes of locating the Alpha Werewolf.

When Eve is making her way across the country in "...And Then There Were None," Bobby mentions that her presence has caused a group of vampires to act up.

Castiel brings Lenore to Bobby's house so that they can ask her for Eve's location. She is hesitant, but eventually tells them where to find Eve: in Grants Pass, Oregon. She then asks to be killed. With Eve on earth, her nest has abandoned her and returned to feeding on humans, and even she has given in to her bloodlust on one occasion. Sam and Dean try to persuade her that after Eve is destroyed things will improve, but Lenore is doubtful. Castiel places a hand on her head and kills her to move things along.

In Grants Pass, Oregon, they find Eve experimenting with creating hybrid monsters. On examination of some of the hybrids in a bar, Dean finds they have strange hybrid attributes, one creature has vampiric teeth in addition to the spike of a Wraith protruding from its wrist. Dean dubs these hybrids Jefferson Starships.

After capturing Red in The Man Who Would Be King, Bobby compliments him on taking down an entire vampire nest and capturing them in Swan Valley. At the same time, Crowley has a captured vampire he experiments upon, discovering that when he burns or impales Eve's brain, it causes the vampire pain because there is still a connection between them even though she is dead.

While in Purgatory, Dean teams up with a vampire named Benny Lafitte, who feeds only on blood transfusions stolen from the blood bank, to find a way out into Earth, along with Castiel. The three make their way through Purgatory heading for a way out that Benny knows that will work for a human. Benny believes he can hitch a ride with Dean as he considers himself a former human.

Later, Dean succeeds in escaping Purgatory with Benny's soul and performs a ritual that resurrects Benny at his gravesite. The two go their separate ways, but Benny later contacts Dean and they agree to not keep in touch for a while, but that Benny can call Dean if he needs help.

Benny, knowing that if his maker gets wind of the fact that he is alive will come after him, goes after his maker "The Old Man" to kill him first. He tries to get a vampire from his former nest named Quentin to tell him where his maker is, but Quentin ambushes him with two other vampires. Benny wins the fight and kills all three vampires, but is badly injured and calls Dean for help. Dean brings Benny blood from Benny's truck to help him recover and Benny explains what he's doing so Dean decides to join him. The Old Man and his former nest are vampire pirates or "vampirates" who ambush private yachts going on long journeys, drain the crew, then sink the boats. Benny fell in love with one of the people he ambushed, Andrea and left his nest for her. Consequently, was killed for it and Benny believes Andrea was killed as well. Benny wants revenge and knows his maker will come after him if he finds out Benny is alive again. Finding the nest on Prentis Island, Benny is shocked to find that Andrea has been turned into a vampire and is captured. Dean escapes and kills all of the vampires in the nest while Benny escapes with Andrea's help, kills a vampire named Sorento and confronts The Old Man who refuses to fight back.

Benny kills The Old Man and is shocked to learn that instead of wanting to completely destroy the pirating operation and start a new life with him, Andrea wants to take it over herself. Benny realizes to his heartbreak that the Andrea he loved is dead and as she goes to attack him, Dean kills her from behind. Benny is left completely distraught and heartbroken and wonders why Dean brought him back when he could've pretended to help. On the mainland, Benny meets Sam and leaves, recognizing that Sam and Dean need to be alone.

A rouge vampire named Desmond decides to create his own nest and tries to entice Benny, who has moved back into his hometown in Louisiana and is looking after his great-granddaughter Elizabeth secretly, into helping him. However, Benny refuses so Desmond warns him that he will kill someone every night until Benny agrees. Desmond's first victim draws the attention of mentally-unstable hunter Martin Creaser who is following Benny and believes him to be the killer. He calls in Sam and Dean, but Dean believes Benny's story even after finding him after he buries Desmond's second victim. Despite Dean's belief, Martin and Sam go after Benny until Dean tricks Sam into leaving. Dean and Benny go after Desmond after Benny pretends to agree to join him to learn where he is and in the confrontation that follows, Benny kills Desmond. However, seeing Dean bleeding from an attack stirs Benny's bloodlust.

Afterwards, realizing that the situation means he can no longer stay there, Benny leaves, but Martin, obsessed with killing Benny, kidnaps Elizabeth to force him to return. In the confrontation that follows, Benny pretends to allow Martin to kill him, but turns the tables on him and in the fight that follows, rips out his throat, killing him, before fleeing.

Benny calls Dean for help after his loneliness starts to get to him. Though Dean, who believes Benny's story after learning what happened from Elizabeth, promises to meet Benny for coffee once the case he is on is over, he later calls Benny to say a final farewell, having decided to cut all ties to the vampire.

In Conway Springs, Kansas, a group of young hunters are killing vampires as they have supposedly killed their families. However, these vampires are revealed to have been newly turned and the true killer, Seth, is working with their mentor Victor Rogers in exchange for being left to roam free so he can create the next generation of hunters. After learning the truth, Krissy Chambers takes Seth down with darts filled with dead man's blood and he is presumably killed. Seth's blood is then used to cure his latest victim who hadn't drank human blood yet and was later, saved.

In Purgatory, a vampire attacks Sam, but after he stabs it in the stomach with Demon-Killing Knife, Sam decapitates him with the vampire's own blade.

After learning from Naomi that Sam got to Hell through Purgatory and that Ajay is dead, Dean goes to Benny for help. Benny agrees to let Dean kill him and then lead Sam and Bobby Singer to the portal out as he doesn't feel like he belongs on Earth. After agreeing to see Benny when Sam returns, Dean decapitates him.

Three more vampires attack Sam and Bobby. Sam kills two, but the last one nearly overpowers Bobby before Benny rips out his throat. Recognizing Benny, Sam lets him lead them to the portal out where three more vampires, enraged at Benny working with a Winchester again, arrive to attack. Having never intended to return to Earth, Benny stays behind to hold the vampires off and is last seen fighting them as Sam escapes.

In Devil May Care, hunter Tracy Bell pretends to be having car trouble and a man who is really a vampire offers her a ride, planning to feed on her. Tracy takes the ride, but overpowers and kills the vampire in the struggle that follows, leaving his headless body and van on the side of the road.

During the events of Slumber Party, Charlie Bradbury later tells Sam and Dean that as one of the hunts she did she killed a teenage vampire.

While hunting down Cuthbert Sinclair during Blade Runners, Sam and Dean are confronted by two vampires from his "zoo" as a test and easily kill them.

In Alex Annie Alexis Ann, a young woman named Alex is arrested in Sioux Falls, South Dakota and is attacked by her vampire "brother" Cody as she ran away from the "family." Sherriff Jody Mills kills Cody and calls in Sam and Dean to hunt the nest and protect Alex who they learn was kidnaped several years before and has been acting as a lure for people who won't be missed for her "family." Feeling guilt for her lifestyle, Alex ran away, but is recaptured by her "mother" Celia and her surviving "brothers." Sam, Dean and Jody hunt the nest down and are all captured, while Alex agrees to be turned into a vampire. Connor and his surviving brother try to drain Sam and Dean of blood, but Dean recovers, kills both and frees Sam before heading off to save Jody. Jody realizes that Celia kidnapped Alex to replace a daughter she lost a long time ago and Celia nearly kills her after Alex refuses to. However, Alex injects Celia with dead man's blood and Jody kills her. As Alex hadn't drank any human blood yet, Sam and Dean are able to cure her and Jody helps her through the ordeal.

During Bloodlines, while visiting a monster club, Ennis Ross is confronted by a waiter named Maurice who is revealed to be a vampire. Ennis tries to kill him with a gun armed with silver bullets to no avail and is nearly killed himself before Maurice is decapitated from behind by Dean. Sam and Dean later explain to Ennis what Maurice was as he'd never encountered a monster before.

In Hibbing 911, a group of vampires led by Starr who consume all of a victim, not just their blood, go to Hibbing, Minnesota in hopes of regaining their former leader, Sheriff Len Cuse. Len covers up their murders of two people in town, but they draw the attention of Sheriff Jody Mills who calls in the Winchesters. After finding Len over one of the vampires victims, the Winchesters, Jody and Sheriff Donna Hanscum track him to an old farmhouse where they are captured by the nest. After Len refuses to kill the humans, Starr kills him, but Dean breaks free and kills Catfish and the other vampire. Star goes after Jody, but is killed by Donna before she can harm her.

During The Werther Project, Dean hunts a vampire nest alone to take some of the edge off of the Mark of Cain. He is able to kill all six vampires single-handedly before Sam even arrives. Also, Benny appears as a hallucination under the influence of the Werther Box.

During Brother's Keeper, Dean is called in on a vampire hunt by fellow hunter Rudy. Dean is able to rescue one of the two victims alive and kill the two vampires involved, but gets Rudy killed in the process. This causes him to realize he's out of control and summon Death in an attempt to die.

In Keep Calm and Carry On, as Lady Toni Bevell describes the British Men of Letters method of operation, they are seen capturing a vampire with dead man's blood after being alerted by wards to his presence and taking him to a warehouse where the vampire is executed by Arthur Ketch.

In LOTUS, Arthur Ketch shows the Winchesters and Castiel a weapon the British Men of Letters created through a combination of technology and magic to use against vampires. As beheadings are inefficient, especially against entire nests, the weapon irradiates vampires, reorders their DNA and makes their own blood toxic to them. The Winchesters are impressed by this method of killing vampires.

In First Blood, while the Winchesters are missing, Castiel notices a case of vampire killings in Lancaster, Missouri and attempts to solve the case by himself. However, Castiel is unable to locate the vampires and runs away after three more young women die. Castiel brings the case to Mary Winchester who offers to let Castiel help her, but he feels he will just get in the way. Mary locates the vampires and kills them all single-handedly just before Castiel calls her to tell Mary that he has heard from Sam and Dean.

In Stuck in the Middle (With You), Mary tells Wally that while she's been working with the British Men of Letters, they have taken out four vampire nests.

In The Raid, the British Men of Letters have begun an effort to exterminate all vampires in the United States, starting in the Midwest. In the course of a month, the British Men of Letters have killed 230 vampires with only eleven being left alive in the Midwest. This draws the attention of the Alpha Vampire who leads an attack on the British Men of Letters compound. After the Alpha Vampire is killed by Sam Winchester with the Colt, the remaining vampires flee.

In Wayward Sisters, Claire Novak compares scars with Kaia Nieves. One scar is from a bar fight with a vampire. Later, after Sheriff Donna Hanscum arrives to help, its mentioned that Donna has killed "a lot of vampires" over her career as a hunter.

In Breakdown, the cashier Marlon is revealed to be a vampire helping Terrance Clegg's operation to butcher people and sell their remains to monsters. Marlon turns Doug Stover into a vampire after revealing his true nature. Doug is subdued by Dean with dead man's blood while Donna kneecaps Marlon with a shotgun and forces him to give up Clegg's location before killing Marlon. As Doug had not yet fed, Dean is able to use Marlon's blood to cure Doug and restore Doug's humanity.

In Beat the Devil, the Winchesters, Castiel and Gabriel rescue two teenagers from a creature that is revealed to be an Apocalypse World vampire. Now starving, the vampires have devolved into more bestial creatures who are ravenous for human blood. With a vampire nest blocking the only way to Mary and Jack's base, the group and the teenagers are forced to pass through. During the trip, one of the teenagers is dragged off while Sam has his throat ripped out by one of the vampires and dies.

Later, Sam is resurrected by Lucifer who is seeking his son. Lucifer is able to use his restored powers to hold back a ravenous horde of vampires in order to force Sam to hear him out and eventually leave with him.

In Stranger in a Strange Land, Sam sends some of his hunters out to deal with a nest of vampires that he receives word is menacing people. Later, he gets a call from Anael that they have a big problem as Michael meets with a vampire, having decided monsters are the perfect creatures to ally himself with.

In Gods and Monsters, Michael performs experiments upon Lydia Crawford's nest using their blood and angel grace in an effort to find a way to enhance the abilities of monsters. Though her nest mates are killed, Lydia Crawford manages to escape and later explains what she saw to Sam, Mary Winchester and Bobby Singer. Michael visits Lydia after they are gone and explains that he allowed Lydia to escape in order to bait a trap. Michael then kills Lydia as she is no longer useful to him.

In The Scar, Michael sends some of his grace-enhanced vampires, now immune to dead man's blood, after the alternate Kaia Nieves who owns a spear that can harm him. Kaia kills the vampires, baffling Jody Mills as tests indicate they are not monsters. Later, more of Michael's vampires attack and nearly kill the Winchesters and Jody, but Dean frees Kaia who uses her spear to kill the vampires.

In Nightmare Logic, manifestations of Apocalypse World vampires appear amongst the other manifestations conjured up by the enhanced djinn Neil.

In The Spear, a vampire named Brett acts as one of Michael's lieutenants in Kansas City. When Sam infiltrates the office building Michael is using as a base to rescue Jack, he kills Brett in the parking garage.

In Nihilism, a female vampire is part of the group of monsters that attacks the Men of Letters bunker to rescue Michael. Along with the other monsters, she is disintegrated by Jack.

In Atomic Monsters, the Winchesters investigate the murder of cheerleader Susie Martin whose body was chopped into pieces after death. Dean determines that they are dealing with a vampire after finding a vampire fang in Susie's corpse, though the Winchesters are confused by the chopping up of the body. Later, another cheerleader, Tori, is kidnapped. Using security footage of the abduction site, Sam is able to identify the kidnapper's car and trace it to Henry Whitman who the Winchesters suspect was attacked and changed into a vampire a few weeks to a month before. In the Whitman's garage, Sam finds and rescues Tori and its revealed that the vampire is Billy Whitman, Susie's boyfriend. After being attacked and turned by an unknown vampire, Billy eventually lost control of his bloodlust while making out with Susie and his parents cut up her corpse to cover up the killing and kidnapped Tori to act as a blood bag for Billy in their efforts to protect their son and give him a normal life. Guilt-ridden and knowing he will lose control again and hurt someone else he loves, Billy has his parents pin the murder and kidnapping on him while the Winchesters, with Billy's full acceptance, take him out into the woods to be killed so Billy can't hurt anyone else.

At the same time, Sam experiences a nightmare where he has become the leader of a demon army after becoming addicted to demon blood. Benny is a part of a force of hunters that tries and fails to stop Sam, resulting in Benny's death.

In The Trap, a Leviathan reveals that Benny Lafitte was torn apart by his own kind after returning to Purgatory. God later explains that Sam's nightmares, including the one with Benny, were of alternate reality versions of the Winchesters.

In the alternate timeline that God shows to Sam, vampires are one of the types of monsters running increasingly rampant across the Earth as a consequence of God getting locked away. Claire Novak is killed by vampires when a nest that she and Jody Mills go after proves to be much larger than expected. Sam and Dean are eventually turned into vampires themselves on a hunt gone wrong before they are hunted down by hunters including Bobby Singer and Jody. The Winchesters kill the other hunters and Dean kills Jody, but Bobby is able to kill Sam. The outcome of Dean and Bobby's fight is unknown, but this timeline is erased from existence when Sam chooses not to trap God.

In The Heroes' Journey, vampires are amongst the spectators at the monster fight club. Later, after capturing the Winchesters, Cutty intends to pit them against the massive vampire Maul. The Winchesters are rescued by Garth who blows up the fight club and all of the monsters inside, but Maul manages to survive. Ultimately, Garth manages to kill Maul by cutting downwards through his head with a machete.

In Last Holiday, after Mrs. Butters brings the bunker out of Standby Mode, the monster radar reveals a vampire nest located 50 miles away. At the nest, the Winchesters find two vampires watching Dark Shadows and complaining about the differences between how they and the vampires on the show live. When the Winchesters burst in, interrupting their show, the vampires get up to face them, but are decapitated in seconds without a fight.

In Carry On, the Masked Vampires target a family in Akron, Ohio, killing Lyle, ripping out the tongue of his wife and kidnapping his sons. When Sam and Dean investigate, they recognize the wife's drawing of the vampires' masks from their father's journal from a hunt he embarked upon in 1986 for a similar string of attacks by masked vampires in Akron, Canton and East Sparta. John was never able to find much, but his journal identifies the vampires as targeting families that are living outside of town and are isolated. By locating the family in Canton that matches that description, the Winchesters are able to kill one of the vampires before they can attack and capture the other. Threatened with a slow death if he refuses to cooperate, the vampire explains that his nest harvests a couple of children every few years, raises them up, feeds them and then eventually "juices" the kids as they don't eat "fast food." In exchange for a quick death, the vampire directs the Winchesters to the nest's location.

At the nest, Sam and Dean find and rescue the children, but are attacked by the four surviving vampires who armed with blades of their own. Sam and Dean each manage to kill one of the vampires before being overpowered. Jenny, the young woman who was turned into a vampire fourteen years before by Kate, encountered the Winchesters on their first vampire hunt and escaped with Kate after the destruction of Luther's nest, appears and is revealed to be a part of the nest as well. Though not the leader, Jenny has called dibs on killing the Winchesters. However, Sam recovers and decapitates Jenny from behind and they engage the two remaining vampires in a fight. Though Sam kills one, the other impales Dean upon rebar sticking out of a wooden post before Sam can kill him too, mortally wounding Dean. Dean dies for the last time of his injuries shortly thereafter before a grieving Sam brings the kids back home.

In Cast Your Fate to the Wind, a vampire cult named Il Soarta searches for the Gem of Ursitoare, which can predict the fate of its holder. Diana reveals that the cult struck a bargain with the Romanian fate gods, the Ursitoare, for the amulet. Its use was sacred until the cult leader attempted to escape his destiny, leading to Il Soarta's loss of the amulet, which then fell into the possession of the Men of Letters. Following a vision foretelling John Winchester's demise, the cult besieges the Men of Letters Clubhouse. Carlos Cervantez exterminates two vampires outside by dousing them with holy water using his long hair, while Mary eliminates another seven in the tunnels. The Il Soarta chieftain invades the vault where John is concealed with his mother. However, John intentionally electrocutes himself to death, poisoning the vampire with dead man's blood as it feeds on him. Weakened by the tainted blood, the vampire leader is slain by Mary, who, with Millie's assistance, manages to resurrect John.