Donkey Kong Country Returns is notable for introducing entirely new antagonists, the Tiki Tak Tribe led by Tiki Tong, instead of reusing King K. Rool and the Kremlings, who were the series' primary villains since Donkey Kong Country. According to the game's producer Kensuke Tanabe, he and Shigeru Miyamoto discussed early in development whether or not to bring K. Rool back, but they ultimately concluded that it was unnecessary, leading Tanabe to ask Retro Studios to create their own new antagonists. The game's senior designer Kynan Pearson claimed that this was done in an effort to differentiate the game from the previous Rare-developed Donkey Kong Country titles. King K. Rool and the Kremlings would eventually return years later as Spoiler:surprise final antagonists in Donkey Kong Bananza. Pearson felt that the character's return was bittersweet for him due to Nintendo's original mandate when he worked there, while also feeling that it allowed for K. Rool's appearance in the game's climax to be more effective.

In the original Wii version of the game, there was a mechanic where defeated enemies would retain their collision for about 25 frames, a window lasting a fraction of a second. In cooperative play, this allowed for a second player to trail behind and quickly use defeated enemies as a platform, allowing for two players to traverse platforming sections without one or the other frequently dying by falling into a pit. However, for unknown reasons, this mechanic was removed in the Nintendo 3DS and Nintendo Switch versions of the game, making the multiplayer gameplay more difficult and unfair in certain sections. This mechanic was later restored in an update for the Nintendo Switch version in 2026.

Retro Studios dev Bryan Walker recalled in an interview how unexpectedly understanding Shigeru Miyamoto was to him and his team's vision for the project in the planning stages and how considerate and mentorlike he was with their pitches and ideas. When the final moments of the collaboration were almost done, however, Walker recalled that Miyamoto said something in English to them that stuck with them forever: "Please take care of DK. He is my friend."

In World 1-1, if the player goes back into the starting hut there will be a television showing static. Using the ground pound move will make the television change to the intro of Donkey Kong Country Returns.

At the end of the level "Lift-off Launch", green lights can be seen in the sky. These lights can be found in northern parts of the world, and go by the name of the "Northern Lights". This suggests that the game takes place in a northern part of the world.

In the level 2-6, the zebra, giraffe and elephant in a boat in the background are possibly a reference to the movie Madagascar.

In 7-R, "Lift-Off Launch", there is a large gyroscope with three spinning rings around a core; this is a reference to the Main Dynamo present in Metroid Prime 2: Echoes.

In honor of the release of the game, Nintendo trademarked the phrase "It's on like Donkey Kong."

In the Options menu, a coral reef is shown that is believed to be based on the Rare logo, possibly to pay homage to the developers of the previous Donkey Kong Country titles.

Donkey Kong Country Returns is the first game in the series to feature no water levels. Water levels were planned for the game, but according to the game's producer Kensuke Tanabe, the old underwater levels "felt slow... It just wasn't the kind of gameplay we were looking for."

In the original game, Donkey Kong will play a Nintendo DS if left idle for a long enough time. In the Nintendo 3DS version, these animations were removed for unknown reasons. In the Nintendo Switch version, the animations were swapped out with the Nintendo Switch idle animations previously used for Funky Mode in Donkey Kong Country: Tropical Freeze.

Mr. Game and Watch from the Game and Watch series makes a cameo appearance in the Foggy Fumes level.

In level 3-2, Button Bash, there's a stone monkey which opens up its mouth for you to go into. The monkey is wearing a tie that somewhat resembles the American flag. This tie is a reference to the tie worn by professional gamer Billy Mitchell, who held the world record for the top score in the Donkey Kong arcade game. This Easter egg remained in all later re-releases of the game despite a legal controversy between 2018 and 2024, where record keepers temporarily removed his Donkey Kong score among others and contested their authenticity.

In the background of the Foggy Fumes level, there appears to be the skull of the mini-boss Crocomire from Super Metroid.

In 2010, UK video game retailers "Game" and "Gamestation" created a promotional offer for the release of the game, where the first 20 customers at select stores could trade in actual bananas for a copy of the game.