After slaughtering Abaddon's Demon Army, the Knight of Hell Cain falls back under the influence of the Mark of Cain. As a result, Cain decides to slaughter his bloodline which he feels is cursed. At some point, Cain obtains this knife which he uses to kill numerous people, including Tommy Tolliver.
After discovering Cain's murder spree, Sam and Dean Winchester along with Castiel and Crowley decide to kill him in order to stop Cain. When Cain goes after Tommy's son Austin Reynolds, he is armed with the knife when Castiel tries to stop him. After Cain is unharmed by Castiel's attack, he telekinetically flings Castiel with a flick of his knife, knocking the angel out briefly. After teleporting into the barn, Cain stabs Austin with the knife despite his and Sam's pleas, only to discover that Austin is actually an illusion created to draw Cain into a devil's trap. Cain kept his knife out while confronting Sam and Crowley, but put it away between when they left and Dean came in to confront him.
During the fight between Dean and Cain, Cain fought unarmed, relying on his combat skills, super strength and telekinesis instead. After being overpowered by Cain, Dean notices the knife in Cain's sheath as Cain prepares to kill him with the First Blade. As Cain swings the First Blade at him, Dean grabs Cain's knife from his sheath and uses it to cut off Cain's right hand, defeating him. Dean holds the knife in his left hand afterwards and as he kills Cain with the First Blade.
After killing Cain, Dean emerges carrying both the knife and the First Blade. Its further fate is unknown. Dean may have kept the knife for himself or got rid of it.