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Nine days after the deaths of Alexander, Marcus, and William in Underworld: Evolution, humans have captured Selene during "The Purge", a global military crusade to exterminate vampires and lycans. Vampires have waged guerrilla warfare against the government, but the humans have overrun them, forcing survivors to hide underground and resist the humans on their own.
Twelve years later, human governments have concluded their campaigns against the vampires and lycans. They have annihilated 95% of the vampire race and believe lycans to be extinct. Selene, dubbed "Subject 1", is freed from cryogenic suspension by "Subject 2" and escapes the medical corporation Antigen. The corporation is ostensibly trying to make an antidote for the virus that creates vampires and lycans. Selene has visions that she follows, believing them to be linked to her lover, the lycan-vampire hybrid Michael Corvin. The visions lead her to a building where she is followed by David, a fellow vampire. While talking with David, Selene has another vision and discovers a young girl, Eve (Subject 2). Eve is revealed to be a hybrid and the daughter of Selene and Michael. Selene and David flee the building with Eve but are attacked by a group of diseased lycans.Eve is badly wounded by one of the lycans, and David insists that they take her to his coven. David's father, Thomas, blames Selene for provoking the destruction of the vampires. Thomas tells her that Michael died twelve years ago. A vampire woman offers her blood to the wounded Eve, who appears unaware of the effects of drinking it. With Selene's encouragement, she accepts the gift and quickly heals.
Jacob Lane, director of Antigen, is revealed to be a lycan along with his son, Quint. The "antidote" the corporation has been working on is a drug designed to make lycans immune to the deadly effects of silver on their species and enhance their physical abilities. Eve's genetic code is required to achieve this, so Lane sends Quint with other lycans to the vampire coven to recapture her. Expecting a human attack, David tries rallying the vampires' resistances to fight back, while his father orders everyone to evacuate and hide. Most of the vampires arm themselves. Unexpectedly, lycans arrive in large numbers, and many vampires are slaughtered, a huge blow to the coven, which now assumes that humans and lycans are working together. Selene encounters a "super lycan", later revealed to be Quint, whose body is changed by injections containing Eve's genetic material. Selene is knocked unconscious during the fight, Eve is turned over to the lycans by Thomas in exchange for their departure, and David is mortally wounded. Selene decides to save Eve but first revives David using the immortal blood given to her by Alexander Corvinus.
Selene enlists the help of Detective Sebastian, a human who tried investigating Antigen. Sebastian agrees to help save Eve, admitting he was married to a nurse who became a vampire. The two of them lived together as husband and wife, but when authorities were going door to door killing vampires and lycans, his wife exposed herself to the daylight rather than suffer at the hands of humans. Selene destroys part of Antigen's headquarters using silver gas explosives, forcing Lane to move Eve out of the building to perform an operation that will create more of the lycan super-serum. Selene escapes the lycans in the building and finds Michael (Subject 0) cryogenically frozen, puncturing his cell with a gunshot to defrost him. Escaping in a van, Lane is intercepted by Sebastian, and then by Selene, who causes the van to crash. Quint arrives and fights Selene.
Eve awakens on her stretcher, freeing herself and fighting Lane, who has injected himself with the super-serum. Sebastian and David aid Eve in her fight and she kills Lane while Selene kills Quint.
Selene attempts to free Michael but finds he has already escaped. Eve, telepathically seeing through Michael's eyes, learns he is on the roof. Selene, Eve and David run to the roof, only to discover that Michael has already gone. In a voice-over narration Selene concludes with her determination to reunite with Michael and take back the world from the humans and lycans.
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