Link made his first right-handed appearance in Twilight Princess for Wii. Up through Twilight Princess for GameCube, Link had traditionally been left-handed, but with the introduction of motion controls, director Eiji Aonuma decided to make him right-handed so it would be more comfortable to "swing" the sword with your right hand (which is how most people were playing). Instead of re-doing Link's model though, the Wii version of the game was simply flipped horizontally, reversing the positions of everything from east to west.

Twilight Princess is one of the few Zelda games that does not have a magic meter. As such, there is no need for Green Chu Jelly, which is an item used to refill magic in other Zelda games. However, in the Wii version, it is possible to obtain Green Chu Jelly through normal means. But it is only possible once, in the "Cave of Ordeals", room 19. Carefully kill all of the purple Chus and let a Blue Chu and and Yellow Chu merge. Kill it and then scoop up the remains with a bottle. Link will now be holding a bottle of Green Chu Jelly. It has no text, description or effect whatsoever. However, performing this trick in the GameCube version produces a Purple Chu instead.

A popular image macro known as "Gaijin 4koma" (JP: "4-frame foreigners"), depicting a group of fans reacting with indifference to something and then excitement, used to compare the meme editor's and/or popular opinions on two particular subjects, consists of two photos taken by IGN of their reporters during Nintendo E3 presentations in the early 2000s. The excited photo being towards the announcement of The Legend of Zelda: Twilight Princess in 2004, while the indifferent photo was taken during the announcement of Pac-Man Vs. in 2003.

While Navi does not appear in Twilight Princess, she has unused lines and sound effects which can be found in the game's data, including those which may have been intended to be heard from the Wii Remote speaker in the Wii version.

According to Eiji Aonuma, The Legend of Zelda: Twilight Princess was inspired by "The Lord of the Rings" movie trilogy, as he wanted the game world to be vast enough to match expectations fantasy audiences had at the time:

The Master Sword being a sword that would get pulled out from its pedestal by those chosen appears to be derived from the Arthurian Legend of Excalibur, also known as the "sword in the stone". Similar to how King Arthur was divinely chosen to be able to pull out Excalibur, Link was given the power by the Goddesses to pull out the Master Sword. Because of these similarities, the Master Sword was renamed to "Excalibur" in the French localizations of The Legend of Zelda: A Link to the Past, The Legend of Zelda: The Wind Waker, and The Legend of Zelda: Twilight Princess.

The character design of Purlo, who runs the STAR game in the thoroughfare of Castletown, was directly based on Tingle. According to Eiji Aonuma, he is what Tingle would look like if he was made with a realistic design.

There is a minigame Link can play with a Cucco in the Hidden Village towards the end of the game called Cat Hunt. The object of the minigame is to find 20 cats hidden throughout the village, and Link will be rewarded with a Piece of Heart. In the English translation of the game, the cats have no names and all say "Meow! What fun, meow!" when caught, but in the French translation they each have personal names and custom dialogue.