Rayne (Kristanna Loken) is a "dhampir", the offspring of a human mother and a vampire father. Her father is Kagan (Ben Kinsley), and Rayne watched him slay her mother when she was about seven years old. Ever since, Rayne has vowed to avenge her mother's death by killing her father. There are others who are also fighting against vampires like Kagan. At some point in the past, Viscount Elrich (Billy Zane) created a secret society called the Brimstone. His daughter Katarin (Michelle Rodriguez) continues to run Brimstone, even though Elrich eventually was turned into a vampire himself. Katarin and her two friends, Vladimir (Michael Madsen) and Sebastian (Matthew David), provide the backbone for the rest of the 30 or so members of Brimstone. They are currently hiding in a fortress somewhere in the Romanian countryside.
Working in a Carnival as a sideshow freak sometime in 18th Century Romania, dhampir Rayne is routinely burned by holy water, stabbed, and fed sheep's blood so that she can heal instantly, all for the amusement of the paying onlookers. When news of her performances gets around, three members of the Brimstone, a secret society of vampire hunters, set out to find Rayne. Unfortunately, Sebastian, Vladimir, and Katarin arrive the morning after Rayne has escaped the Carnaval, so they are forced to pick up her trail and keep searching. Rayne has joined a group of travelers, who drop her off in a nearby village where she is summoned by a fortune teller (Geraldine Chaplin) who tells her that her father is Kagan, the most powerful vampire in the land. Rayne reveals that Kagan raped and later killed her mother, and asks the fortune teller how to find and kill him. The fortune teller tells her about "the eye," a talisman which Kagan is seeking. If Rayne could get the eye, which is kept in the Solambrium Monastery, she would be sure to get an audience with Kagan.
Rayne doesn't know it, but Kagan has also been searching for her. He's had one of his vampire henchmen, Domastir (Will Sanderson), pick up her trail. Domastir has overheard what the fortune teller told Rayne about the eye. When he relays the message to Kagan that Rayne is going in search of the eye, Kagan orders that Rayne be watched and, when she does get the eye, she be killed.
Rayne heads for the monastery, followed by both the three Brimstones and by Domastir with his band of about a dozen thralls. The monks give Rayne dinner and a place to rest for the night. While everyone is asleep, Rayne sneaks around looking for the eye. She kills a deformed monk in order to get the key around his neck, uses the key to open the chamber where the eye is kept, dodges rotating blades to get to the pedestal where it sits, but the moment she picks it up, the doors slam shut and the room begins to fill with holy water. She leaps to the ceiling where she hangs upside down to escape the water. While waiting, she looks at the eye, which is suddenly assimilated into her own eye. Suddenly, the chamber door opens up, revealing the head monk (Udo Kier). He tells Rayne that the eye has made her impervious to water. The eye is actually one of three talismans -- an eye, a rib, and a heart -- that belonged to a long-dead vampire named Belial. Belial found a way to protect vampires against water, sunlight, and crosses. Upon his death, his body parts were scattered in fear of some vampire getting hold of them and becoming invincible. Because the eye has now become apart of Rayne's body, the monk won't let her leave the monastery.
Suddenly, a shout rings out that the monastery is being attacked. Domastir and his thralls begin knocking off monks right and left. Sebastian, Vladimir, and Katarin join the fight, but it is Domastir who succeeds in knocking out Rayne and riding off with her body. Katarin is ordered to return to Brimstone to tell them that Domastir has Rayne and that Rayne has the eye, while Sebastian and Vladimir pursue Domastir. Because day is approaching, Domastir is forced to take shelter at Leonid's (Meat Loaf) palace of prostitutes, vampires, and other decadents. When Leonid lays eyes on Rayne, he wants to add her to his collection, but Domastir says that Rayne is for Kagan. However, when Leonid learns that Rayne has the eye, he has Domastir placed in chains and means to get the eye from Rayne even if he has to cut it out himself.
Sebastian and Vladimir have infiltrated the palace. They come upon Rayne just after she's been stabbed several times by Leonid. Vladimir tosses a blanket over Rayne and shoots out the windows, allowing in the sunlight. Leonid burns to death, and Sebastian and Vladimir carry Rayne's body back to Brimstone headquarters. They place Rayne in a cell, but Rayne promises not to feed from then and asks only for animal blood so that she can heal and get on with her business. A few rats later, Rayne is healed. Vladimir convinces her to stay and train with Brimstone so that they can attack Kagan together. A new set of clothes and a fresh sword, and Rayne is ready to go.
Although Rayne's stay at Brimstone seems to have given morale a shot in the arm, Katarin isn't pleased by Rayne's presence. In her correspondence with her father, the Viscount Elrich, the two of them express the belief that Brimstone is lost, although Elrich continues to protect the secret location of the Brimstone Society in order to protect his daughter's safety. On the other hand, Elrich is plotting to unseat Kagan. Meanwhile, Kagan has obtained the rib talisman and has ordered his thralls to comb the land for Brimstone's location, destroy everyone in Brimstone, and bring Rayne to him.
After arming themselves with gunpowder, swords, and holy water from an arms dealer in town, Vladimir, Sebastian, and Rayne return to Brimstone. Before they reach the fortress, however, they are met by a wounded patriot. With his dying breath, he reveals that Katarin has betrayed them and that everyone in Brimstone is dead. That night, while Vladimir and Sebastian sleep out under the stars, Rayne tries to sneak off on her own. When Sebastian wakes up and tries to stop her, Rayne confesses that it is because of her that all those people died and that she's going to go it alone from now on. Also, she intends to find the heart talisman because she thinks that will give her the fighting edge against Kagan.
Rayne's first stop is back at the Brimstone fortress, where she finds everyone is either dead or dying. Suddenly, she hears voices and hides behind a wall. It is Domastir telling his thralls that the damphir's body is not among the victims and that they must return to tell Kagan. Rayne goes looking deeper in the fortress and comes upon Katarin looking for the heart, which she tells her companions was buried by her grandfather at the bottom of a pool of water. Katarin dives into the pool to retrieve it, and Rayne follows. Katarin stabs Rayne in the abdomen, grabs the talisman, and begins swimming to the surface. Rayne follows. When they reach the surface, Rayne grabs Katarin, slits open her neck, and drinks her blood, which immediately heals Rayne's wound. As Katarin lies dead, Rayne now has the heart and she leaves to confront Kagan.
Fearing that Rayne is no match for Kagan, Vladimir and Sebastian saddle up their horses and ride to Kagan's castle, where they hide in the bushes, waiting for Rayne to show. When she does, she rides straight to the gate and calls out, "I am here for Kagan." Rayne is immediately escorted to Domastir. She gives him the box with the heart in it and asks to see Kagan, but Domastir tosses her in a cell in the dungeon until it comes time to remove the eye. He then takes the heart to Kagan. In order to get inside the castle, Vladimir and Sebastian storm the front gate and are immediately captured by the guards and placed in a cell near Rayne.
It is time for the ceremony that will remove the eye from Rayne and unite it with the heart and rib. Rayne is escorted to the ceremonial chamber and tied to an altar while they ready the knife. Meanwhile, Vladimir and Sebastian have tricked a thrall into entering their cell, overpowered him, and managed to escape. They storm the ceremonial chamber and start whacking away at the guards. Sebastian cuts Rayne free. Meanwhile, Kagan has opened the box which holds the heart and found it empty. He runs his sword through Vladimir and then turns on Rayne. They cross swords several times, managing to stab each other, but Kagan ends up on top. As he prepares to cut out Rayne's eye, Sebastian tosses Kagan a bottle of holy water, then shoots an arrow into it. The bottle breaks and the holy water eats away at Kagan's face. Rayne takes the opportunity to thrust an arrow through Kagan's heart and he turns to dust. With Kagan and Vladimir both dead, Rayne runs to Sebastian. He is also severely wounded. Rayne offers to turn him, but he refuses and dies in her arms.
Rayne, the sole survivor amid the carnage, takes her seat on Kagan's throne.