The movie opens in Sola Mira Hive, where Priest (Paul Bettany), Priestess (Maggie Q) and Black Hat (Karl Urban) hunt vampires. They are caught in a trap, and Black Hat is captured by vampires.
An animated story gives the background - man vs. vampire, destroying each other and the land. Man withdrew behind the safety of the church, and are defended by priests, men with extraordinary abilities. After the vampires were defeated, the priests were sent back into the general population, no longer needed.
Owen Pace (Stephen Moyer) lives on a farm in the wasteland with his wife Shannon (Madchen Amick) and daughter Lucy (Lily Collins). The land is arid, but he has hopes that the radiation levels will soon be low enough to plant crops. Lucy is resentful of their sheltered existence, and during dinner, vampires attack. Owen is hurt and Shannon is killed, and Lucy screams.
In the futuristic Cathedral City, church messages from Monsignor Orelas (Christopher Plummer) are broadcast to the public, as Priest walks through the crowds. He goes to confession, but the advice is a recording. On the way home, Hicks (Cam Gigandet) tells Priest his brother Owen was attacked. He goes on to say that he's going after the vampires, and asks Priest to help him. Priest goes the church to ask to go after the vampires. Orelas doesn't believe that it was vampires, and warns Priest not to question the church's faith. Priest breaks his vow.
Lucy is imprisioned on a train, and Black Hat is her captor. Priest arms himself and hops on a futuristic cycle to travel to the wasteland. He visits his brother's place, and meets up with Hicks. They go to see Owen, and he asks Priest to recsue Lucy and kill all the vampires.
Meanwhile, Monsignor Orelas sends a posse of priests after Priest to capture or kill him.
Priest begins to track the vampires. They arrive at a vampire reservation, and find the guards gone. Priest and Hicks enter the underground hive and find the guards, dead. Hicks is attacked by familiars, and Priest takes them apart. The sun goes down, and the vampires emerge. They fight them off, and a familiar tells them Lucy has been taken west, to the Sola Mira Hive.
An informant tells Black Hat about Priest and Hicks, and Black Hat turns him.
Priest and Hicks arrive at the Hive, which brings flashbacks to Priest. He leaves Hicks at the entrance, and moves deeper into the hive. Along the way, he finds the Priestess, and they battle a hive guardian. Hicks helps them kill it. They track the vampire army to the town of Jericho, where the Black Hat's train is. Black Hat's army is killing the inhabitants, and the rest of the chuch posse tries to stop him.
By the time Priest, Hicks and Priestess arrive, the town is deserted. The other priests are crucified, and Prist realizes the vampires are headed to the city. They plan to head off the train, stop the vampires, and rescue Lucy. Priestess and Priest share a moment, and she gives him a special cross.
On the way to the train, Hicks learns that Lucy is Priest's daughter. Priest and Hicks get on the train, and Priestess drives ahead to blow the tracks. On top of the train Black Hat confronts Priest, and tells him how he became a human vampire - through the blood of the Queen. Black Hat is now stronger than Priest.
Priestess fights off familiars on motorcycles, but one of them has destroyed the detonator for the explosives.
Hicks fights his way through vampires, and finds Lucy. Priest joins him, and they pursue. Black Hat attacks and knocks Hicks off the train. Hicks finds a motorcycle and gives chase. Black Hat nails Priest to a wall and sets him on fire.
Priestess arms the explosives and drives her motorcycle to the train. Meanwhile, Priest arms himself with her special cross and throws it at Black Hat. He rescues Lucy, and Priestess' bomb destroys the train. Hicks arrives and finds Priest holding Lucy. She realizes Priest is her father, and even Priestess has survived.
Priest returns to the church and throws a vampire head at the feet of Orelas. He tells Orelas that the war is just beginning, and rejoins Priestess in the hunt for vampires. End.