Struggling to find his place in the world while juggling school and family, Brooklyn teenager Miles Morales is unexpectedly bitten by a radioactive spider and develops unfathomable powers just like the one and only Spider-Man. While wrestling with the implications of his new abilities, Miles discovers a super collider created by the madman Wilson "Kingpin" Fisk, causing others from across the Spider-Verse to be inadvertently transported to his dimension.

After reuniting with Gwen Stacy, Brooklyn’s full-time, friendly neighborhood Spider-Man is catapulted across the Multiverse, where he encounters the Spider Society, a team of Spider-People charged with protecting the Multiverse’s very existence. But when the heroes clash on how to handle a new threat, Miles finds himself pitted against the other Spiders and must set out on his own to save those he loves most.

Taking place right after the events of Spider-Man: Across the Spider-Verse, Gwen Stacy leads the newly reformed Spider-Gang in a race against the Spider-Society, led by Miguel O'Hara, to find Miles Morales, who is trapped in the home dimension of the spider that bit him, along with an alternate version of himself as the Prowler, while the Spot begins his deadly attack in his dimension with far-reaching multiversal consequences.