ASCII text reading "ICE KEY" is present in the ROM, a remnant of when Donkey Kong 64 was planned to be part of the Stop 'n Swap feature in several Rare games.

Early screenshots of Donkey Kong 64 showed that Donkey Kong's Treehouse originally had a shower stall in it with a poster of Banjo and Kazooie on it.

Grant Kirkhope, composer for the game, provided the voice of Donkey Kong.

Despite being an E-rated game, the intro got away with mild swearing. Near the end of the song, the last line during Chunky Kong's portion of the song says, "But this kong's one hell of a guy". The game was developed by Rare, a British company, and in Great Britain the word isn't considered offensive. In later versions of the song the word is replaced with "heck".

The xylophone that appears in Candy's Music Shop is the same xylophone that Mumbo plays in the intro to Banjo-Kazooie.

In Donkey Kong's cabin hangs a picture of a dolphin. This is a reference to the code name of the Nintendo Gamecube, "Dolphin". The game was released before the Nintendo Gamecube was revealed, and is thought to be the earliest reference to the code name "Dolphin."

The Kong's weapons were originally real guns, as opposed to the comical fruit and nut based weaponry seen in the final game. This was changed in response to Shigeru Miyamoto being horrified by them upon paying a visit to the Rare headquarters to check on the game's development. He then sketched out and offered a replacement that would be more fitting to the world of Donkey Kong, which would ensure that the game would receive a child friendly rating.