In the Japanese and non-English European versions of the game, Vivian is portrayed as in some way gender non-conforming, with the original Japanese script, like a lot of pop culture addressing gender identity at the time, being ambiguous as to whether Vivian is a transgender woman being referred to in an unflattering light, or a cisgender male crossdresser. A rough translation of the Japanese text for Vivian as a party member reads:


This was changed in the English localization to make Vivian a cisgender woman with "a bit of an inferiority complex." The Italian version of the game instead appears to portray Vivian as a proud transgender woman:

In the X-Naut Fortress there is a changing room. If you drop into the room from the vent above, both you and your partners will turn into 8-bit sprites, with the original Super Mario Bros music playing in the background. You can turn back to normal by exiting the room.

All of the partners from the first Paper Mario were originally going to make cameo appearances in this game, but were scrapped for unknown reasons. However, Parakarry and Lady Bow do remain in the game, and make appearances at the beginning and in Poshley Heights after completing the game, respectively.

The enemy character Red Bones' name in Japanese is "Buraddi Honenoko", which translates to "Bloody Son of Bone" (or "Bloody Dull Bones"), which could hint to a Koopa Troopa having met a gory demise, then being resurrected as a Dry Bones, but still had a stained shell.

When Peach starts up Sir Grodus' computer, the opening sequence featuring is actually the startup animation from the Famicom Disk System. The yellow disk also resembles one of the FDS's game disks.

At one point, the parrot below Creepy Steeple might say "Shine get! Shine get!", a reference to the phrase in the Japanese release of Super Mario Sunshine.

One of the crows in Twilight Town talks about opening a real estate website called "Luigi's Mansion," but his friend warns him against possible copyright infringement.

Lord Crump's theme has the sound of Pac-Man (Atari 2600) dying mixed into it.

A small toad in Petalburg will rave about his favorite video games. First he will talk about Fire Emblem for the Game Boy Advance. Later he'll be talking about Paper Mario for the Nintendo 64. Finally, he'll tell you that he's playing Paper Mario: The Thousand-Year Door itself, and states that the ending is worth playing the game all the way through.

The house in the back alley of Rogueport's main square originally had a toad-shaped chalk outline and dried blood on the floor. These signs of violence were removed from the U.S. version of the game.

In the German version, the Snow World is called "Großfrostheim" which translates into "Great Frost Home". This, however, is a reference to the German city "Großostheim", which is where the Nintendo of Europe headquarters are located, and where the game was localized.

In the Spanish version, Rawk Hawk is named Hawk Hogan as a reference to Hulk Hogan.