All the puppies are following each other and moving in a pack, except for the white one, who's playing with a squeaky carrot toy. An 8-year-old girl named Penny comes into the store and buys the white puppy, giving it a collar with the name tag 'Bolt'. Five years later, 13-year-old Penny and Bolt are in the park, when Penny receives a phone call from her father (who turns out to be a television actor, pretending to be Penny's father) telling her that "something's come up at work" and he won't be home for a while. As he warns Penny not to go back to the house, the evil Dr. Calico sneaks up on him.

Before he's interrupted, Penny's father tells her not to worry, that Bolt will protect her. A clip-montage plays, showing that her father (a scientist) has genetically altered Bolt, giving him a variety of superpowers, as well as the distinctive black lightning-bolt mark on his flank. Back to the story, Penny uses a pair of enhanced binoculars to spy on one of Dr. Calico's associates, who is speaking to him via a hologram video. Dr. Calico says that he knows how to make Penny's father reveal his secrets and that they just need to find a "certain thing" first. Calico's associate leaves the building, and Penny and Bolt follow at a distance. They follow him down an alleyway, at which point he disappears...and then a car is seen racing towards them. Bolt runs head-on toward the car, which is flung over his head and lands upside-down behind him upon impact. Calico's associate is still alive but trapped in the car, and Penny has Bolt hold the car (with his teeth) over the side of a bridge in order to make him tell them where Calico is.

Calico is evidently in Bolivia, but by now, they've been found again. Penny pulled out a fold-out scooter and zoomed off down the highway, with Bolt running by her side as a group of helicopters and motorcycling bad guys (armed with electrified gloves) chase them.