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For generations, a secret war has been waged between vampires and Lycans, an ancient species of werewolf. Selene, a Death Dealer, a vampire specialized in assassinating Lycans, tracks two Lycans. Selene's motivation goes beyond duty; she also wants revenge because, 600 years ago when she was a child, Lycans slaughtered her family. The vampires believe they defeated the werewolves many centuries ago and killed their leader, Lucian, and that they must kill the surviving Lycans. Selene locates the Lycans' den and learns they have developed a new kind of UV bullet capable of killing vampires.
Later, Selene arrives at the mansion of her vampire coven and, recounting these events, urges an attack on the Lycans; but the vampire regent, Kraven, dismisses her proposal. Selene determines that the pair of Lycans may have been following a human, Michael Corvin, and continues her investigation. In a Lycan lair, a scientist named Singe tests blood from kidnapped descendants of the ancient Corvinus family, trying to find a pure source of the ancient and powerful Corvinus bloodline. Soon after Selene finds Michael, the pair are attacked by Lycans, including their leader, who is revealed to be Lucian. Lucian bites Michael, but Selene helps Michael escape and the two become attracted to each other.
Selene discovers that Kraven was the only witness to Lucian's supposed death. Sensing a conspiracy, she awakens a dormant powerful vampire elder, Viktor, who has been hibernating. Viktor is angered by his early awakening, as it was Marcus' turn to rule the coven. Viktor believes Kraven and orders Selene to acquiesce to the vampire hierarchy. Kraven meanwhile has secretly planned to kill the third vampire elder, Amelia, and her companions, with Lucian's help.
Selene informs Michael about the feud and her past. She then binds him, fearing that he will kill innocent people after he changes into his Lycan form at the coming night's full moon. Selene captures Singe while Michael is captured by Lycans. Selene returns to the mansion with Singe, who admits to Viktor that the Lycans are trying to combine the bloodlines of the two species with the Corvinus Strain, the shared source of both the Vampire and Lycan bloodlines, to create a powerful hybrid. After Kraven flees the mansion because Singe has revealed his cooperation with Lucian, news arrives that Amelia, who was coming to awaken Marcus, has been killed by Lycans. Viktor kills Singe and tasks Selene to kill Michael.
While Michael is being held captive, he learns from Lucian that Viktor's daughter, Sonja, and Lucian were lovers. Lucian tells Michael that the Lycans were slaves to the vampires. When Viktor learned that his daughter had been impregnated by a Lycan, he killed her to prevent any crossing of the two species, which led to the war. According to Lucian, such a crossing would create a hybrid creature that would be more powerful than both the vampires and Lycans.
Selene arrives with a group of Death Dealers to kill the Lycans and Kraven for his treachery. Kraven shoots Lucian with a newly designed bullet that kills by poisoning a Lycan's bloodstream with silver nitrate. Selene rescues Michael, who is shot by Kraven. Kraven tells Selene it was Viktor who killed her family. Lucian stabs Kraven in the leg and convinces Selene to bite Michael, transmitting the vampire virus into his bloodstream. Selene complies, and Kraven flees after killing Lucian. Viktor arrives and admits that he killed Selene's family, though he believes she should be grateful as he spared her and made her immortal. He reveals the truth of his love for Sonja - that his choice to have her killed was to protect the vampires and that he must now kill Michael to protect the vampires. Selene eventually kills Viktor with the assistance of Michael, who is now a hybrid. Selene and Michael leave the Lycan lair, now enemies of both species. At the mansion, blood from Singe's corpse seeps into the sarcophagus of the sole remaining Elder, Marcus, a carrier of the original Corvinus Strain.