Early in development, there was going to be an enemy named "Tramplin' Stu", another iteration of Stu in the game. The Tramplin' Stu was supposed to spawn Swoopin' and Strollin' Stu behind it as it walked. To defeat a Tramplin' Stu, you must fill it with water so that the outer shell pops, then you stomp on the eye on top of it. The data for Tramplin' Stu can be found in a test map where it shows up correctly, but without its shell. Tramplin' Stu shows up correctly with its shell when moved to the Delfino plaza map.

The layout of the beach in the Sirena Beach stage strongly resembles the GameCube controller. One of the pools represents the grey analog stick, while the others are the A, B, X and Y buttons. The flame at the center is the START button, and the chairs are where the Nintendo GameCube logo would be. The two cabana huts are positioned to form the D-pad and C analog stick.

Within one of the bell towers in Delfino Plaza is a Rocket Nozzle box. The box is inaccessible without the use of a cheat device, and is only visible through the use of camera glitches.

The option to turn off closed captions during cutscenes does not appear in the Japanese release, likely due to them having extensive English voice acting in all versions of the game.

Each location on Isle Delfino contains an Italian word. This is likely in reference to Mario's nationality.
• Delfino Plaza - Dolphin Plaza
• Bianco Hills - White Hills
• Ricco Harbour - Rich Harbour
• Gelato Beach - Ice Cream Beach
• Pianta Village - Plant Village
• Noki Bay - Shell Bay
• Pinna Park - Fin Park
• Sirena Beach - Siren Beach
• Corona Mountain - Crown Mountain