Peter Parker (Tobey Maguire) ha conseguido finalmente equilibrar la balanza entre su devoción a Mary Jane (Kirsten Dunst) y sus deberes como superhéroe. Pero una tormenta se avecina en el horizonte. Cuando su traje se transforma, convirtiéndose en negro y potenciando sus poderes, Peter también se ve cambiado. Bajo la influencia del traje, aparecen en Peter el orgullo y el exceso de confianza, y así comienza a dejar de lado a los que más se preocupan por él. Aunque dos temibles villanos, Hombre de Arena (Thomas Haden Church) y Venom (Topher Grace), consiguen un poder sin parangón y sienten la sed de castigo, la mayor batalla de Peter se libra, no obstante, dentro de sí mismo. Spider-Man tendrá que volver a descubrir la compasión que le convierte en lo que es: un héroe.

Peter Parker has finally managed to piece together the once-broken parts of his life, maintaining a balance between his relationship with Mary-Jane and his responsibility as Spider-Man. But more challenges arise for our young hero. Peter's old friend Harry Obsourne has set out for revenge against Peter; taking up the mantle of his late father's persona as The New Goblin, and Peter must also capture Uncle Ben's real killer, Flint Marko, who has been transformed into his toughest foe yet, the Sandman. All hope seems lost when suddenly Peter's suit turns jet-black and greatly amplifies his powers. But it also begins to greatly amplify the much darker qualities of Peter's personality that he begins to lose himself to. Peter has to reach deep inside himself to free the compassionate hero he used to be if he is to ever conquer the darkness within and face not only his greatest enemies, but also...himself.