Three years after his previous defeat by Crash Bandicoot, Dr. Neo Cortex returns to the Wumpa Islands to exact revenge on Crash and paralyzes Coco to keep her from interfering as with previous attempts. Dr. Cortex dons a Coco-inspired disguise that lures Crash to the bay where he forces Crash into battling his gigantic mech, the Mecha-Bandicoot controlled by Dr. N. Gin. After destroying the Mecha-Bandicoot, Cortexes hoverboard breaks causing him to fall and hit crash into a cave. when they land Cortex becomes enraged and assaults Crash, throwing them down deep into the mining shafts where Cortex originally found the first Power Crystals. The duo are forced to work together to escape the mine before it caves in due to large ant-like beings invading the area in search of crates filled with Wumpa Fruit. After narrowly escaping, Crash and Cortex encounter a pair of odd, anthropomorphic turquoise parrots who proclaim themselves as "The Evil Twins", who have come to destroy the Wumpa Islands. They threaten Cortex with stealing his brain and scare him enough to make him flee. Cortex pleas for Crash's help and finds himself being attacked by bees, which Crash helps in guiding him through a series of traps created by the native tribesmen and their leader, Papu Papu for hunting purposes. Cortex runs into Papu Papu, who then punishes Cortex by tying him to a totem pole with a Power Crystal resting on the top. Crash breaks Cortex out and narrowly escapes from the angry tribe when he meets with a local Wumpa fruit farmer. The farmer has troubles with large worms eating his saplings and is promising the duo a Power Crystal if they can help get rid of the creatures. Cortex instead paralyzes the farmer and steals the crystal, but are forced to get rid of the worms anyway in order to continue on from the farm. The Evil Twins once again attack the duo, this time bringing about a totem deity to destroy them. It is learned that they come from the 10th Dimension and that they are also siphoning the good traits from the 1st Dimension to leave it dark and desolate with their machine called the 'Vice-Versa Reversa Ray'. Cortex concocts a plan that he and Crash travel to his new lair near the Antarctic Circle to use the 'Psychetron,' a machine that will allow them travel to the 10th Dimension.
Crash and Cortex attempt to enter the Iceberg Lair through the front entrance, but the remote key refuses to work and they are forced to go the long way through a cavernous glacier filled with machinery. They inadvertently free Uka Uka in the process, who is enraged by Cortex's continuous failures and attacks them using black magic to form into an ice creature. Uku Uka is convinced by his brother Aku Aku to help him defeat the Twins, however, they are easily defeated by the evil duo and Crash and Cortex are ambushed by more robotic ant creatures sent by the Twins. After beating them back and learning that Cortex is responsible for the Twins' rein of terror (and learning the Twins own a generous amount of treasure), the Aku-Uka brothers reason out that if they deliver Cortex to the Twins, they may just spare the Wumpa Islands. Despite this, Crash instead decides to work with Cortex and defeat the Twins and steal their riches, influenced by Cortex's plight. They reach the Psychetron only to find they do not have enough power in order to make the quantum leap. Cortex begins to lament but then realizes that N. Gin owns a battleship which can take them to another continent to find more Power Crystals. They reach as far as an observation deck, but cannot make it on foot in time before N. Gin sets sail. Crash gets an idea and uses Cortex as an impromptu snowboard to comedic effect. After wiping out, they form a large snowball which destroys Dingodile's winter home. Among the wreckage, Dingodile catches wind of Cortex's plot to steal the Twins treasure and goes to tell several other adversaries of Crash's, including Doctors Nitrus Brio and Nefarious Tropy. Crash enters the battleship alone and infiltrates the airborne crow's nest where he is forced to battle N. Gin as he launches rockets and TNT crates at the marsupial. After destroying the crow's nest, it crash lands into the battleship and sends Crash down a hatch just outside the galley kitchen. The battleship chef, Rusty, chases after Crash in an attempt to use his meat for recipes. Crash escapes, though is inadvertently attacked by N. Gin, who lands directly on a TNT crate surrounded by the entire surplus of explosive crates. It explodes, causing the ship to sink and launch Crash onto a far away ice sheet where Doctors N. Brio and N. Tropy attempt to get info out of Crash on the whereabouts of the treasure. This attempt fails due to Crash's initial muteness and Dr. Nitrus Brio transforms into a giant frog-like monster to attack Crash. After the two doctors' defeat, Crash returns to the iceberg lair with Cortex and they get to the Psychetron with Crash's newly collected crystals found on the battleship.
Cortex begins to initiate the Pyschetron with their newfound power supply, but encounters Coco, who believes he's kidnapped Crash. Coco kicks Cortex, which sends the two crystals he had into the machine and damaging it and paralyzing Coco again in the process. In order to repair it, Cortex needs to find the one person who helped him build it, Nina Cortex, his mischievous cyborg niece. Cortex and Crash travel to Madame Amberly's Academy of Evil, a private school for the rich and evil, and make their way into the school via the boiler room. Dingodile has also made his way to the school and interrogates Cortex to no avail. He then duels with Crash and is subsequently knocked out, not to be seen again. Crash and Cortex discreetly make their way through the gloomy halls of the school and find Nina, convincing her to leave the private school and help them fix the Psychetron. After escaping from a floating school bus and encountering Madame Amberly, it is revealed that the Evil Twins are actually Cortex's former pet parrots Victor and Moritz, mutated by the 'reverse-radiation' present in the 10th Dimension. Once Nina fixes the Psychetron (by banging on the control panel), she travel to the 10th Dimension with his uncle and Crash, finding it to be a bizarre and gloomy mirror universe of Earth. Crash's 10th Dimension's villainous version, Evil Crash, kidnaps Nina and takes her to his desolate home on Twinsanity Island. Crash is forced once again to use Cortex as a snowboard and they corner Evil Crash. Cortex valiantly sacrifices himself in Nina's stead which leads to a chase scene. After they escape Evil Crash and rescue Nina, they make their way to the Twins' compound overrun with their ant-like minions. Along the way, they find the fabled treasure and also discover N. Brio, N. Tropy and N. Gin had made it there first. Cortex shows disinterest in the treasure and scolds the villains for attempting to steal it, claiming that Spyro the Dragon "may want his gems back". Spyro punishes the villains by breathing fire at them while Crash, Nina and Cortex carry on to find the Twins. Nina and Neo work together to weaken the Twins' Mecha-Parrot by destroying its weapons, while Crash ultimately defeats the Evil Twins by using a repaired Mecha-Bandicoot to destroy the Mecha-Parrot. The evil twins flee to Evil Crash's home only to be devoured by Evil Crash himself. After returning to their dimension, Cortex tries using the Psychetron to get rid of Crash, claiming his existence was a constant reminder of his worst mistakes, but it once again malfunctions and instead sends Cortex into Crash's brain where multiple miniature personifications of Crash dance for Cortex's amusement and chagrin.
In the post-credits scene, Crash and Cortex have been undergoing dual therapy. As the day's exercise, their therapist (never seen on screen, only heard) asks Cortex to closes his eyes and fall backwards for Crash to catch as their first trust exercise. Cortex at first does not agree with the exercise as he does not like, let alone trust Crash and also has basophobia. Eventually, he decides to go through with the exercise, however Crash is temporarily distracted by a fallen apple, diverting his attention from the trust exercise and causing Cortex to fall onto the floor. Cortex then grumbles that he "hate bandicoots" before falling onto his back dizzily as Crash consumes the apple.
- Lex Lang as Doctor Neo Cortex, an evil scientist who teams up with his arch-nemesis, Crash Bandicoot.
- Debi Derryberry as 8-year-old Cortex
- Derryberry also voices Coco Bandicoot, the younger sister of Crash Bandicoot.
- Debi Derryberry as 8-year-old Cortex
- Mel Winkler as Aku Aku, the guardian of the Wumpa Islands and the father figure of Crash Bandicoot and his friends.
- Mel also voices the Tribesmen.
- Michael Ensign as Doctor Nefarious Tropy, a henchman and old friend of Uka Uka and the self-proclaimed master of time.
- Susan Silo as Nina Cortex, the gothic niece of Doctor Neo Cortex. She doesn't speak much until the ending when she says, "Huh?"
- Silo also voices Madame Amberley, the headmistress of the Academy of Evil, and the former teacher of Neo and Nina Cortex.
- Quinton Flynn as The Evil Twins, Victor and Moritz, two parrot twins owned by Doctor Neo Cortex until they're turned into villains in the 10th Dimension.
- Flynn also voices Doctor N. Gin, the right-hand man of Doctor Neo Cortex.
- And the penguins.
- Alex Fernandez as Uka Uka, the evil younger twin brother of Aku Aku.
- Alex also voices Farmer Ernest, an mutated emu farmer who runs an orchard of Wumpa Fruit tress in the outskirts of N. Sanity Island.
- Dwight Schultz as Dingodile, the ally of Doctor Neo Cortex.
- Dwight also voices Papu Papu, the obese chief of the native tribe of N. Sanity Island.
- And Rusty Walrus, a large blue walrus that served as the chef of N. Gin's battleship.