Cast :
- Kate Beckinsale - Selene. After the events of Underworld: Evolution, she becomes the first Vampire-Corvinus Strain Hybrid, with superior physical abilities to an average Vampire and can walk in sunlight along with other abilities she has yet to discover, including a telepathic link with her daughter Subject 2/Eve. She is the lover of Michael Corvin and mother of Eve, whom she conceived with Michael in the second film.
- Sandrine Holt - Lida, a Human scientist working for Antigen. Hired to look after Subject 2/Eve, Lida sees her - a surrogate daughter, and help develops Subject 2/Eve's adolescent mind in Selene's absence by schooling her in various forms of education since infancy, seemingly without Lida's superiors' knowledge.
- India Eisley - Subject 2/Eve, who is only referred to as Subject 2 in dialogue. She is a Hybrid and daughter of Selene and Michael and the latest descendant of Alexander Corvinus. Like her father, Eve has all the abilities of Vampire, Lycan, and Corvinus clans along with additional abilities including a telepathic link with her parents, making her unique yet also an outcast among them. A scientist from Antigen, Lida, is the only person Eve knew who showed her compassion, and educated Eve in secret during her isolation within the facility. Despites Lida's kindness, Eve desires a loving relationship with her mother, eventually leading her to free Selene from her cryogenic chamber. The Lycans intend to use her and her father's DNA to make themselves immune to silver and to have the Vampire's ability to heal faster.
- Theo James - David, a Vampire; son of Thomas and an ally of Selene. He brought Selene back to the coven in hope she would train some of the members as Death Dealers so that they could execute an offensive against both Humans and Lycans, as there aren't any left. He also has great admiration for Selene.
- Michael Ealy - Detective Sebastian; he was once married to a Vampire, thus leading him to become Selene's ally. He witnessed his wife burned by sunlight when the government conducted a door-to-door extermination. Knowing not all Vampires and Lycans are evil, Sebastian helped Selene rescue Subject 2/Eve. In the process he inadvertently discovers a conspiracy within the company, led by rogue Lycans, to dominate the human, vampire, and even their own races.
- Charles Dance - Thomas, a Vampire Elder; Father of David and the surviving coven's acting leader. He is aware of Selene's past, and, as such, does not trust her, becoming unhappy when David brings her to the coven.
- Stephen Rea - Dr. Jacob Lane; the head scientist of Antigen. Secretly a Lycan-Corvinus Strain Hybrid and the father of Quint. Working to perfect the Lycans so that they can destroy the Vampires and rule over the Humans.
- Kris Holden-Ried - Quint Lane. Son of Dr. Jacob Lane and the first test subject of Antigen. Dr. Lane faked Quint's death in order to keep his experiments on him a secret after Quint was discovered to be a Lycan years before, one result is that he is now immune to silver.
- Robert Lawrenson[4] - Waterfront Cop
- Lee Majdoub - Desk Guard #1
- DJ Miller - Lycan #1
- Ron Wear - Jack Fletcher
- William Francis - Police Officer
- Daniel Boileau - Lab Worker #3
- Panou - Old City Cop #1
- Adam Greydon Reid - Alan
- Julia Rhodes - Antigen Tech
- Jeff Sanca - Truck Driver
- Benita Ha - Surgical Nurse
- Christian Tessier - Guard
- Jesse Branden Dahl - Vampire #1
- Marvin Duerkholz - Leroy
- Wes Bentley - Scientist at Antigen
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