In the game, the player follows and controls the super dog Bolt and Penny on various missions taking place in the fictional TV show universe from the movie. The levels are shared between Bolt and Penny. By using Bolt's superpowers, the player can fight hordes of enemies. New powers, such as Superbark and Laser Eyes, will be unlocked during the game. While Bolt has superpowers, Penny has only her maneuverability by using her Wheelbar and is incentivized to take down enemies in a stealthy way. The game focuses on Bolt's fake TV life, not the actual movie storyline. Penny's father has been kidnapped by the evil Dr. Calico, and Penny and Bolt must travel through 5 countries to rescue him.
Penny's father has been captured by Calico and his organization in an attempt to construct a powerful weapon of untold power. Penny and Bolt travel to Italy in an effort to find out more, but they get nowhere other than learning of Calico's location in Belize.
Calico finally manages to capture Penny once she and Bolt find his temple base in the Yucatán jungle. To prove to Penny's father that she is alive and being held hostage, Calico gives Penny a cell phone with her father on the other line. Calico then attempts to flee the crumbling temple with his new captive but decides to leave her there with Bolt to die, saying, "As long as the professor believes she's alive, he will do what is asked and finish the weapon. Too bad we can't all have nine lives".
However, Penny and Bolt survive and discover Calico's new base in the Russian Arctic with a large rocket in the center. But upon entering, Penny notices micro-focusing mirrors and realizes that it is her father's satellite instead of a warhead. All too late as Calico seals the entrance and, to Bolt's horror, launches it with Penny inside screaming for help. Bolt then jumps onto the rocket and dismantles it in mid-flight, crashing it into the snow.
The two then venture to Calico's harbor in China (discovered by Penny by hacking into a computer system), where they discover a ship full of warheads. Just then, Calico arrives at the harbor, enraged to find Penny still alive, and even more so as he watches his prized freight ship sink to the bottom, thanks to Bolt, who then pursues Calico, escaping in his car to a nearby train station. He arrives at the station and boards his train, closely followed by Penny and her dog. Just as the duo finally corners the doctor, he slips into a hidden room and escapes on his private jet, though not before Penny secures a homing beacon onto the hull, leading them right to Calico's base.
Penny and Bolt trace his jet back to his main base, where Penny's father is being held. Upon arriving, Calico tests his new weapon on Bolt, draining him of his powers and strength and capturing Penny, due to her emotions getting in the way of the mission. Once Penny is inside, he explains his plan to use Penny's father's micro focusing mirror-equipped satellites to bounce a beam around the Earth and neutralize all atomic weaponry except for his own, meaning he can nuke anyone, when he wants, how he wants. As he leaves, he violently kicks Bolt's cage proving Penny of her limited knowledge. During this, Penny maneuvers her way out of her hand bindings.
Once free, she attempts to find her father to save Bolt, but is only captured by Calico once again, just as Bolt regains his powers and Penny sabotages the control panel, raising the bridge so Bolt could reach her. Once he does, Penny escapes from her guard captors yet again and launched a pre-prepared, rocket-powered cart at the main power generator (which Bolt exposed). Then, using what power remains in the generator, Calico duels with Bolt, using powerful lightning bursts. After the lengthy and hard-fought battle, Bolt finally defeats him. Beaten and weakened, he attempts to escape on his private helicopter, launching numerous missiles as a decoy, just as Penny finally finds her father (aboard the helicopter). While Bolt tries to apprehend Calico by grabbing his foot, Penny's father pleads to her and Bolt to allow Calico to escape with himself to stop the missiles, reminding her that she had the power to find him again. Regretfully, Penny orders Bolt to stop the missiles as Calico escaped with her father.
The game closes with an epilogue informing the player of Penny's continuing battle. Then, depending on the version, you either get a "tune in next week" style message or cut to Rhino's dialogue.