In an attempt to kill the Knight of Hell Abaddon, Dean Winchester tracks down Cain in hopes of gaining the First Blade, the only weapon that can kill a Knight of Hell such as Abaddon. During their encounter, Cain gives Dean the Mark of Cain which he needs to power the First Blade before teleporting Dean and Crowley to safety and then smiting an army of demons sent after him by Abaddon. During this encounter, Cain tells Dean that after he kills Abaddon, he will one day have to kill Cain himself as well.
After getting the Mark from Cain, Dean kills Abaddon and briefly becomes a demon after being killed by Metatron before being returned to humanity by Sam. Still possessing the Mark and falling further and further under its influence, the Winchesters and Castiel begin a search for Cain in hopes that he will be able to give them a cure for the Mark.
After killing Abaddon's demon army, Cain falls back under the influence of the Mark himself and its bloodlust. Believing his bloodline to be tainted due to the Mark and the number of killers his bloodline has produced, Cain begins slaughtering his descendants, killing dozens of people and burying them in a mass grave. His murder of serial killer Tommy Tolliver draws the Winchesters attention while Castiel locates Cain's burial site where Cain himself explains the purpose behind his actions to Castiel. Knowing that Castiel will tell Dean what he has seen if he lets him go and Dean will bring the First Blade into battle against him, Cain departs without harming Castiel.
As predicted by Cain, Castiel informs the Winchesters of their encounter and Dean becomes determined to kill Cain to prevent the genocide of millions of people in Cain's quest to extinguish his bloodline. Sam is able to determine that Tommy has a 12-year-old son named Austin Reynolds in Ohio that is most likely Cain's next target. Needing the First Blade, Dean calls in Crowley who possesses the weapon and together they devise a plan to trap and weaken Cain once he arrives so that he can be killed.
Once prepared, the Winchesters, Castiel and Crowley wait for Cain outside of the barn on the farm where Austin Reynolds lives. As predicted, Cain eventually shows up as Austin leaves the barn and prepares to kill him. Before he can, Castiel steps in and while Sam escorts Austin to safety, faces off with Cain. Castiel blasts Cain with white light, but the powerful demon is completely unharmed by the angel's attack. Cain then disarms Castiel and flings him through the side of a nearby truck. With Castiel out of the way, Cain follows Sam and Austin towards the barn and attempts to knock down the door. Failing that, Cain teleports into the barn behind Austin and stabs him, only to have Austin dissolve into a purple cloud. Cain quickly realizes that Austin was actually an illusion spell cast by Crowley to lure him into the barn while the real Austin is safely away. More amused than anything, Cain discovers that the illusion successfully lured him into a devil's trap painted on the barn floor and covered in hay to trap and weaken him. While Cain informs Sam and Crowley that a devil's trap can't hold him for long, Sam informs him that it won't need to before the two leave to where Dean is waiting outside.
With Cain successfully ensnared, Dean tells the others to stay outside and prepare to deal with whatever comes out, afraid that fighting Cain will result in him losing control of the Mark of Cain. The others promise to do so and Crowley hands over the First Blade after Dean tells him that if he comes out and doesn't give it back, they'll have a bigger problem to worry about. Dean then enters the barn where Cain has cleared the hay off of the devil's trap and he greets Dean who recognizes the futility of trying to talk Cain down. Cain insists that he's doing the world a favor by killing off his bloodline and challenges Dean to kill him if he thinks he can.
Dean enters the devil's trap and attacks Cain, however, Cain is so powerful that he retains a measure of his incredible strength even in the devil's trap and proves to be a much better fighter than Dean who is unable to land a blow with the First Blade as a result. Eventually Dean manages to land a few punches, but fails to cause any real harm to Cain who is surprised by the lack of strength Dean is showing when armed with both the Mark and the First Blade. Cain realizes that Dean is holding back in hopes that if he doesn't succumb to the Mark, he will exit the fight the same as he entered it. Cain insists upon his belief that it is impossible to resist the Mark, that there is only remission and relapse, using himself as an example. Cain continues to prove to be too strong for Dean to beat even with the devil's trap binding most of Cain's powers and Dean tries reminding Cain that when they first met, Cain had told him that Dean would one day have to kill him. Unconcerned, Cain uses telekinesis to fling Dean through a nearby window, causing Dean to drop the First Blade.
As Dean recovers, Cain explains how he let Castiel go so Dean would bring the First Blade into battle and Cain could finally get it back. Cain is able to retrieve the First Blade from the floor with telekinesis and restrains Dean one-handed as Dean tries to stop him from getting it and throws him to the ground. Cain tells Dean that he's saving Dean from the terrible fate Cain suffered by killing him, stating that Dean would kill Crowley, then Castiel and then Sam with varying reactions until Sam's murder turns Dean into as much of a savage as killing Abel did to Cain. As Cain speaks, he holds the First Blade to Dean's throat and Dean notices Cain's knife as he does. Stating "your welcome, my son", Cain raises the First Blade to kill Dean, but as he swings, Dean grabs Cain's knife and cuts off his right hand, disarming Cain and effectively defeating him.
Standing, Dean grabs the First Blade while Cain asks him why he hesitates. Dean pleads with Cain to tell him that he can stop killing, but Cain resolutely tells him that he will never stop. As Cain bows his head in an apparent acceptance of his fate, Dean plunges the First Blade into Cain's back with a cry, killing him.
After killing Cain, Dean rejoins his friends outside and gives Castiel the First Blade rather than Crowley as he promised. Dean then admits to lying both about Cain targeting Crowley and giving the First Blade back afterwards, causing Crowley to start to distrust the Winchesters again. On Dean's instruction, Castiel hides the First Blade somewhere while Sam states to Dean that if he can kill Cain without losing control of himself, there's still hope for him. However, Sam privately admits to Castiel that Dean is in trouble, recognizing that Dean is losing more and more of himself as time goes on despite their best efforts.
With Cain's death, the Knights of Hell officially become extinct, with the rest killed by both Cain and Dean, and Dean himself being cured.
- This battle features the first tactical use of a devil's trap when the Winchesters, Castiel and Crowley use one and an Illusion Spell to weaken Cain enough to give them a fighting chance against his incredible power.
- During the battle, Cain displays a partial immunity to a devil's trap when he still retains at least some of his super strength and telekinesis while battling Dean within one. On previous occasions, demons are shown to be completely bound by devil's traps. Cain, while amused by the trap, also stated that it wouldn't hold him for long, not realizing that that wasn't the point of the devil's trap.
- This battle features the final use of the First Blade on the series.
- Unlike in his battle against Abaddon, Dean doesn't rely completely on brute force and the power of the Mark of Cain to win. Instead, he holds himself back to retain his humanity and relies on tactical thinking as well as his fighting skills.