Beam Me Up, Scotty!: A meme circulating around the internet features a picture of the train passengers lifting Spider-Man's unconscious body with the caption, "carefully, he's a hero", even though no such line is actually said in the movie.
California Doubling: The movie was filmed partially in New York City and mixed in with filming in Los Angeles (look at the traffic lights to distinguish NYC scenes from Los Angeles scenes). The train fight between Spidey and Doc Ock had to be filmed in Chicago as that city still has extensive elevated trains running downtown, whereas Manhattan got rid of all their downtown and midtown elevated lines by the 1950s.
Development Gag: The film has a couple of playful jabs at Tobey Maguire's back problems, which nearly forced him to drop out. This includes the "I'm back, I'm back! My back, my back!" scene, and a Freeze-Frame BonusBugle headline claiming link between back pain and brain shrinkage.
Alfred Molina lost some weight for the role of Doctor Octopus, considering he had had to gain some for the part of Diego Rivera in Frida. He said he wanted Doc Ock to have the build of a "1950s weightlifter".
Doc Ock doesn't become fused with his arms until the subway fight.
Doc Ock is obsessed with Spider-Man because the chip he gave him can stabilize his fusion with the bionic arms.
Aunt May would've been angrier with Peter when she finds out that he could've stopped Uncle Ben's killer. Later on, they have a discussion where feeling crushed by your responsibilities is normal, not something you can escape from, and not a special destiny.
Doc Ock captures Spider-Man, and Mary Jane and Harry find out about Peter's secret identity.
And lastly, it's Mary Jane who convinces Otto to abandon the experiment through their prior connection, and Spider-Man breaks free and saves Mary Jane.
Doc Ock aside, previous drafts included the Lizard and Black Cat villains. Another draft featured a younger Octavius who created the spider that bit Peter. In the end, all ideas were scrapped.
An early storyline idea that was thrown out was for Harry to put a price on Spidey's head.
An additional fight scene between Spider-Man and Doctor Octopus on a collapsing water tower was storyboarded but never used in the finished film.
There was to be a scene where Otto, after having become Doctor Octopus, confronts Dr. Connors and ends up attacking him. The scene is used in the video game adaptation, however.
After Spidey quits and J. Jonah is given the discarded suit there was a scene where he prances around in it in his office. This was cut and later readded as a deleted and extended scene because he was supposed to come across as a total goof, and he does, but J. K. Simmons looked too good doing it.
Hey, It's That Sound!: As odd as the end result is, the lizardmen mentioned below (the ones that appear on the fight arena) reuse the damage grunts of a "The Skulls", a street gang that acted as the main enemy faction of the first 3 levels of the previous game.
What Could Have Been: The 2003 trailer sported a more cel-shaded style, and showcased the Lizard, who never made it into the game (though there were lizard men of sorts in the game, though only in the unlockable bonus fight arena mode). One of the hint markers makes a reference to this; "Everyone knows that lizards only live in sewers anyway."
Governor's Island was likely meant to be explorable, as it's name is still on the map. It instead ended up being Dummied Out.
There's a row of doors inside the Daily Bugle, which, when approached, play the "door locked" sound effect that usually plays only when a normally accessible location is temporarily locked.note Non-player stores during nighttime, story locations until (and sometimes after) the location becomes relevant) It's speculated that they were to have some sort of extras behind them, but we will never know for sure.
The Original Darrin: Unlike the first and third games, Kristen Dunst reprises the role of Mary Jane in this game.