In New York City, teenager Miles Morales is struggling to live up to the expectations of his parents, particularly his father, a police officer who sees Spider-Man as a menace. Miles' life changes dramatically when he is bitten by a radioactive spider in an abandoned subway station and gains powers similar to Spider-Man's. While grappling with his new abilities, Miles inadvertently witnesses a battle between Spider-Man and Wilson Fisk, a crime lord known as Kingpin, who is using a supercollider to access parallel universes. During the confrontation, Spider-Man senses that Miles is like him and promises to teach him, but the battle ends with tragic consequences, leaving Miles with a sense of responsibility to stop Fisk's dangerous experiment.

Miles soon discovers he is not alone when he encounters Peter B. Parker, a jaded Spider-Man from another dimension. Peter is older, out-of-shape, and reluctant to fight crime again, but he sees potential in Miles and decides to mentor him. As they work to disable the supercollider, they're joined by other Spider-People from various dimensions, including Gwen Stacy as Spider-Woman, who are also trying to return to their own timelines before their presence causes dangerous anomalies. Together, this diverse group of heroes must overcome their differences, learn to work as a team, and confront their own personal challenges to save the multiverse from catastrophe.