Peter Parker develops super-spider powers after being bitten by a genetically altered spider. The game opens with an optional tutorial, narrated by Bruce Campbell, where Peter learns to use his powers and the player learns the controls of the game and how to read the HUD. After competing in a wrestling match as Spider-Man, Peter is devastated when his Uncle Ben is killed by the leader of the Skulls gang. Peter uses his new powers to track down and defeat the murderer. Peter vows to use his powers for good and to fight evil in Manhattan. Meanwhile, Norman Osborn and OsCorp are investigating the appearance of this new hero. Anxious to develop his Human Performance Enhancer "Super-Soldier" Serum, the main goals of which are already exhibited by the super-hero, Osborn orders the capture of Spider-Man, deploying hunter-killer robots for this purpose. Spider-Man fights these robots and emerges victorious. Meanwhile, the Shocker has just robbed a bank, and is driving away with his thugs as Spidey lands, only to be hit by Vulture. Spider-Man goes after Shocker first. His battle with Shocker leads him through Grand Central Station, into the sewers and to a subway station, where Spider-Man defeats him in a climactic battle. Afterward, Shocker tells Spider-Man about Vulture's lair: an old clocktower on the Lower East Side of town. Spider-Man climbs Vulture's tower, but Vulture escapes. Spider-Man defeats Vulture atop the Chrysler Building.
Osborn's scientists tell him that now two individuals with arachnid DNA are at large in Manhattan, and Osborn orders the capture of both. Spider-shaped robots pursue a desperate Scorpion through the sewers. Peter goes down to the subways to take pictures of his battle site with Shocker, when he runs into Scorpion. After Spider-Man helps Scorpion defeat the machines, the apparently paranoid and unhinged Scorpion turns on Spidey, and they fight. Spider-Man wins, but the Scorpion escapes. Meanwhile, Norman Osborn is fired from OsCorp and takes his own untested super-soldier serum to become the forever-feared Green Goblin. Spider-Man defeats the Goblin at the yearly OsCorp Unity Day Festival. Later, Goblin confronts Spider-Man again and is defeated, but tells Spider-Man of bombs planted downtown. Spidey races against time to defuse the bombs, and is attacked again by Green Goblin, this time with deadly small devices called razor bats.
In the Xbox version only, Norman hires Kraven the Hunter after the bomb threat to capture Spider-Man; having lured Spider-Man in, Kraven poisons the web-slinger with a lethal gas, forcing Spider-Man to track Kraven through zoo corridors filled with traps before he finally confronts and defeats Kraven in a cage match in the main area of the zoo.
After studying a wing of a felled razor bat and determining that it was manufactured by OsCorp, Spider-Man goes to OsCorp to research its connection with the Goblin. He discovers that the company is producing highly dangerous chemical weapons, and shuts down the operation. After finding out that the Goblin is after Mary Jane, Spider-Man chases him down to a bridge. Here, Spider-Man has his final battle with the Green Goblin, who is impaled by his own glider. Spider-Man unmasks the Goblin to reveal Norman Osborn's face. Osborn's last words are, "Tell Harry, I'm sorry." Spider-Man replies, "I'm sorry, too." Mary Jane and Spider-Man are reunited, they kiss. Peter narrates the end of the story as the camera pans out, and he breaks the fourth wall saying "Looks like you're done here. Go outside and play."
- Peter Parker / Spider-Man (voiced by Tobey Maguire)
- Norman Osborn / Green Goblin (voiced by Willem Dafoe)
- Mary Jane Watson (voiced by Cat O'Conner)
- Harry Osborn (voiced by Josh Keaton)
- Herman Schultz / Shocker (voiced by Michael Beattie)
- Adrian Toomes / Vulture (voiced by Dwight Schultz)
- Mac Gargan / Scorpion (voiced by Mike McColl)
- Mendel Stromm (voiced by Peter Lurie)
- Sergei Kravinoff / Kraven the Hunter (voiced by Peter Lurie)
- The Narrator (voiced by Bruce Campbell)
- J. Jonah Jameson (voiced by Jay Gordon)
- Bone Saw McGraw (voiced by Jay Gordon)
- Security Guard (voiced by Dan Gilvezan)
- Bone Saw (In the training mode "Basic Combat")‡ - This character is not present on the Game Boy Advance version.
- Dennis "Spike" Carradine‡
- Herman Shultz/Shocker‡
- Adrian Toomes/Vulture‡
- Mac Gargan/Scorpion‡ - This character is not present on the Playstation 2 version.
- Sergei Kravinoff/Kraven the Hunter‡ (Xbox and Game Boy Advance versions only)
- Norman Osborn/Green Goblin‡
- Oscorp Super Mech‡
- OsCorp Lite Mechs
- OsCorp Super Soldiers
- OsCorp Guards
- Skull Gang Thugs
- Shocker's Thugs
‡ The character is a Boss.