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.
Most of the movie split filming between New York City and Los Angeles (look at the traffic lights to distinguish New York City scenes from Los Angeles scenes).
The train fight between Spidey and Doc Ock was filmed in Chicago, as that city still has extensive elevated trains running downtown, whereas in New York City, most of the elevated lines in Manhattan had been replaced by the subways by the 1950s (with elevated trackage only being prominent in the outer boroughs).
Development Gag: There are 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 Bonus Bugle headline claiming link between back pain and brain shrinkage. That headline is actually a lot more mean-spirited than it seems: Maguire's back problems were a point of contention on set, and it was generally assumed by most of the production staff that he was using his condition to avoid showing up on the set or working on days that he didn't feel like it (regardless of whether or not that was true). At one point, they even threatened to fire and replace him.
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".
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." In addition, unused lines from Spidey indicated that Kraven the Hunter and Calypso were set to appear as part of a Lizard subplot, but got cut. Calypso would remain in as a bonus boss, while the Kraven and Lizard subplot was saved for Spider-Man 3.
Governor's Island was likely meant to be explorable, as its 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.
Unused voice lines suggest one of the "concerned citizens" missions involved Spidey finding items people lost, from stereos to rings, and even trophies.