During a fight against the supervillain Mysterio, Spider-Man shatters a very powerful artifact known as the Tablet of Order and Chaos, creating a rift among the Marvel universes. Madame Web then comes to Spider-Man and explains that she has made contact with three other Spider-Men from three different dimensions to tell them that they must work together to recover the pieces of the tablet in order to restore balance to the worlds. The four versions of Spider-Man are as follows:

• Amazing Spider-Man: The Spider-Man most recognizable due to his famous red and black spider suit. Uses his webbing powers to take down enemies. Resides in the Amazing Universe.
• Noir Spider-Man: This Spider-Man lurks in the shadows of a black and white colored 1930's New York City. His abilities lies in the realm of using stealth tactics and using the shadows to his advantage. Resides in the Noir Universe.
• Spider-Man 2099: This Spider-Man of the future had his DNA mixed with a Spider-Man in an attempt to kill him. This gave him a bunch of futuristic powers in a world ruled by corrupt corporations. Resides in the 2099 universe.
• Ultimate Spider-Man: Clad in the dangerous purple symbiote suit, this Spider-Man is the youngest of the bunch. The suit gives him great power in fighting evil. Resides in the Ultimate Universe.

Each Spider-Man uses their own unique abilities to save their universe from danger. New abilities and power-ups can be unlocked by completing combo challenges found in the Web of Shadows. Fighting is done by doing actions such as punching and kicking opponents. Enemies can be blinded by a webshot and be beaten by special attacks. Each universe contains four supervillains from the Marvel Universe to be fought, all leading up to a final battle against Mysterio. These villains include characters like Sandman, Green Goblin, and Hammerhead.

There are also options to view character biographies, concept art, and cutscenes that you have watched in the game. Three alternate costumes are available for purchase for each Spider-Man.