In an emulator, spawning a Goomba and moving it in the path of a jumping Cheep Cheep will normally cause the Cheep Cheep to hit and kill the Goomba, despite these two enemies never normally encountering each other in the game. However, in World 1-1, spawning a Giant Tail Goomba in front of the rivers at the start of the level to try and get the Cheep Cheep to kill it will instead cause the Tail Goomba to fly off-screen as it has a preset path it travels and attacks on instead of just following Mario/Luigi wherever he walks. Off-screen, it will perform its Tail Swing attack that will mysteriously cause the level itself to take damage, effectively "killing it" and forcing the level to erase almost its entire memory and be collected as one coin, including Mario/Luigi. The game then disables you from quitting or restarting the level.

According to Kenta Motokura, the Tanooki-tailed Bowser is actually a Goomba using a Super Leaf. This was done as an homage to the original Super Mario Bros. game.

According to Koichi Hayashida, Cape Mario was originally considered as a power-up for the game before Tanooki Mario was ultimately chosen.

After the release of the game, the animal rights group PETA created a website and Flash game called "Mario Kills Tanooki." The game involves a skinless raccoon dog chasing Mario, who is wearing the suit. According to Eurogamer, Nintendo later made a statement saying:

"Mario often takes the appearance of certain animals and objects in his games. These have included a frog, a penguin, a balloon, and even a metallic version of himself. These lighthearted and whimsical transformations give Mario different abilities and make his games fun to play. The different forms that Mario takes make no statement beyond the games themselves."

PETA later stated that the game was "tongue-in-cheek", and intended to draw attention to real issues.

Unused audio of a Magikoopa Laughing can be found within the game's data.

Two alternate HOME screen jingles can be found in the game's files. The first is a shortened version of the one used in the final game, and the second is the first 6 notes of the Super Mario Bros. theme. The latter was reused in Animal Crossing: New Leaf for when the player puts on Mario-related clothing.

You can make any dandelions blow away and collect any coins that were hiding by blowing into the 3DS microphone. You can also blow away any ink on the screen left by the Inky Piranha Plants.

It's not possible to fly in Tanooki form, but during the credits, Mario and the Toads appear to be flying while wearing the Tanooki Suit.

While the ending of the Bowser's Message fanfare from Super Mario 64 is used when Mario is shocked by what one of the flying letters has, there's also the first half of the fanfare left unused in the game.

Despite not appearing in-game, a sound effect for Yoshi from Super Mario World can be found in the game's files. In the pre-release trailers, the sound can be heard when Mario jumps on a note block.

Following the events of the massive earthquake that devastated Japan in March 2011, the development of Super Mario 3D Land shifted focus entirely. Nintendo's primary goal became making a game that could bring happiness to a tragedy-struck country.

If enough time passes at the end of World 4-4 and at the beginning of World 8-4, a ghost appears.

World 5-2 is designed in a Legend of Zelda format, using a top-down view, lots of rooms, and no stompable enemies. Also, if you light all of the torches in a certain room using a Fire Flower, a secret door opens and a Zelda sound effect is heard. The level was designed in honor of LoZ's 25th anniversary, hinted at by the level number: "5-2" is "25" backwards.

In World 1-3, if you look up with the binoculars and wait, a UFO will fly by.

In 2010, Nintendo held a competition in Flipnote Studio themed around the Mario series' 25th anniversary, and released an animation for a song with lyrics titled "The Mario Drawing Song" to promote it. This song would later be re-arranged as a slower instrumental and included as the background music that plays on the World Special-8 map in Super Mario 3D Land.