Teenager Miles Morales has difficulties settling into his new boarding school and living up to the expectations of his father, a policeman. One evening, while spraying graffiti in an abandoned subway shaft, he is bitten by a robot spider and subsequently develops spider-like abilities. When Miles later returns to the location, he finds the Collider, a particle accelerator built by Wilson Fisk, aka Kingpin, to penetrate parallel universes.

Miles witnesses a battle between the original Spider-Man and Kingpin's people, the Green Goblin and the Prowler. Spider-Man notices Miles and realizes that he too has spider powers. In the fight, however, Spider-Man is knocked into the collider and badly injured. Before he dies, he gives Miles a USB stick with which the collider can be deactivated and warns him that the machine could destroy the city if it is reactivated. Miles watches in horror as Kingpin kills Spider-Man.

After Spider-Man's death, Miles tries out his new abilities, but damages the USB stick. At Spider-Man's grave, Miles meets Peter B. Parker, an older, worn-out version of Spider-Man from another universe, who has also entered Miles' reality through the activated collider. It turns out that the Collider's operation is causing various Spider-People from other dimensions to manifest in Miles' world, including Gwen Stacy, Spider-Woman; Spider-Man Noir; Spider-Ham; and Peni Parker. They all suffer the effects of the dimensional shift. Miles discovers more of his powers, such as the ability to emit electric shocks and become invisible. The Spider-People realize that the Collider is a danger to all dimensions and must be stopped.