- George Clooney as Seth Gecko
- Quentin Tarantino as Richard "Richie" Gecko
- Harvey Keitel as Jacob Fuller
- Juliette Lewis as Kate Fuller
- Ernest Liu as Scott Fuller
- Salma Hayek as Santánico Pandemonium
- Cheech Marin as Border Guard / Chet Pussy / Carlos
- Danny Trejo as Razor Charlie
- Tom Savini as Sex Machine
- Fred Williamson as Frost
- Michael Parks as Texas Ranger Earl McGraw
- Brenda Hillhouse as Hostage Gloria Hill
- John Saxon as FBI Agent Stanley Chase
- Marc Lawrence as Old Timer Motel Owner
- Kelly Preston as Newscaster Kelly Houge
- John Hawkes as Pete Bottoms (liquor store cashier)
- Tito & Tarantula as The Titty Twister House Band
Two brothers Seth and Richard "Richie" Gecko, wanted by the FBI and Texas police for a bank robbery that has left several people dead, stop at a liquor store with the intent of just picking up a state map, but the psychotic Richie kills Ranger Earl McGraw and the cashier and burns the store down. During the gunfight, Richie is shot in his left hand.
The Geckos are heading towards Mexico, where a contact has arranged a safehouse for them. Along the way they stop at a motel and it is revealed that they had been keeping a bank teller in their car trunk as hostage. While Seth goes out to buy some food, Richie brutally rapes and murders the teller, which infuriates Seth when he returns.
Meanwhile, Jacob, a pastor who is experiencing a crisis of faith, arrives at the same motel with his daughter Kate and his adopted son Scott. Realizing that the family's RV can be used to get them across the border, the brothers kidnap the family, making a truce that if they can make it past the border, Jacob and his family will not be harmed. When the RV reaches the Mexican border, the Gecko brothers hide in the bathroom with Kate, to ensure Jacob will not say anything to the cops. But when one cop comes up to the window to speak with him, Jacob behaves nervously, and says it just him and his son crossing the border. The cop hears a noise in the back and asks what it was. When Jacob replies it was his daughter, the cop comes aboard. He knocks on the bathroom door and Kate answers. When they open the door, they see Kate sitting on the toilet, the brothers hidden behind the shower curtain. Kate yells for them to get out, and the cops leave, believing they have cleared the RV of any suspicious activity. After successfully reaching Mexico, they arrive at the "Titty Twister", a strip club in the middle of a desolate part of Mexico, to meet their contact Carlos at dawn.
Inside, Seth and Richie drink heavily, encouraging the entire group to do the same. Richie takes special notice of the club's star performer, Santánico Pandemónium during an extended solo performance, after which Chet Pussy and some others approach the group, looking to settle the score with the Geckos. In a short confrontation, Richie is stabbed in his already wounded hand, and seeing his blood, Santánico attacks him, undergoing a transformation into a horrific vampire. Chaos ensues as the employees, the strippers and the house band are all revealed to be vampires. Most of the patrons are quickly killed. Richie is bitten by Santánico and bleeds to death. Only Seth, Jacob, Kate, Scott, a biker named Sex Machine and a Vietnam veteran trucker named Frost survive the attack. They quickly establish an alliance in order to survive through the night. Seth convinces the group that Jacob is their best weapon, if he rediscovers his faith, and they also figure out how forming a cross with two objects helps fend off the vampires.
The slain patrons, including Richie, reawaken as vampires, forcing the group to kill them all. During this second struggle, one of the vampires bites Sex Machine's arm. While the group is listening to Frost recount an event from his days in Vietnam, Sex Machine transforms into a vampire and bites both Frost and Jacob before being tossed through a boarded up window, which allows many other vampires, in the form of bats, to enter. Seth and the Fullers retreat to a storeroom and improvise anti-vampire weapons from supplies left by prior victims of the bar. The four stage their final assault on the vampires, their weapons proving effective in destroying many of the creatures. During the battle, Sex Machine is killed by Kate and Jacob slays the vampiric Frost. Jacob transforms, but Scott is hesitant to kill him and gets bitten. He manages to dispatch his father and is dragged down by other vampires. Kate follows the wishes of her brother and kills him, also destroying his attackers.
As the sun rises, only Seth and Kate remain alive, surrounded and low on ammunition. Just then, sunlight breaks through the bullet-holes in the bar walls and burns the vampires. Seth and Kate shoot out more holes, which allows them to survive until Carlos and his guards show up. They open the doors, and the sunlight reflects on the bar's disco ball, killing the rest of the creatures while Seth and Kate flee outdoors. Safely outside, Seth confronts a bewildered Carlos, who asks if the bar was run by psychopaths. Seth sarcastically replies that psychopaths don't explode in sunlight. Angry over the deaths of Richie, Jacob and Scott, Seth demands that Carlos lower his 30% take for his stay in El Rey and telling them to wait in the bar, to which Carlos reluctantly agrees. Kate offers to accompany Seth, but fearing for her safety, he declines and gives her some cash.
After Kate drives the RV away, the camera pans back to reveal that the "Titty Twister" bar is the top of an Aztec temple partially sunk into a valley wall, with many abandoned motorbikes and trucks seen around it which means many people met a horrible end.