Following the January 20, 2016 update, the note within the game's files urging players to wait at least a year before posting sprites online was replaced by a new note reading "you know what i HATE? that's bepis THE TASTE the smell the texture hey.... your drooling......".

This is a reference to a man in Moonside from EarthBound who hates the food the player likes. Creator Toby Fox is a fan of EarthBound, and Undertale was heavily influenced by it. The word "bepis" itself is a slang term for "penis" that originated as a mispronunciation of the word and later evolved into an online meme.

The tracks "Bonetrousle" and "Heartache" were originally composed for an early idea for Deltarune (whose gestation predated that of Undertale) before being repurposed here. "Bonetrousle" was initially the main battle theme for Deltarune before being replaced with "Rude Buster", while "Heartache" was originally titled "Joker Battle", implying that it was initially the battle theme for Spoiler:Jevil before being replaced with "THE WORLD REVOLVING".

The Spoiler:Amalgamate enemies appear to take after an identically-named series of foes from The Halloween Hack, a 2008 ROM hack of EarthBound that Toby Fox made in high school; like the ones in Undertale, Spoiler:the Amalgamates in The Halloween Hack are comprised of multiple earlier enemies fused together.

Furthering the connection to the EarthBound fanbase, Spoiler:"Amalgam", the battle theme for most of Undertale's Amalgamates, is composed mostly of samples from the Cave of the Past and final boss themes in EarthBound, including the processed sample of the Beach Boys' 1970 song "Deirdre".

The Japanese version of the game redubs Flowey's "That's a wonderful idea!" voice clip to a Japanese equivalent. In a DM sent on Twitter to VTuber Houshou Marine, Undertale creator Toby Fox confirmed that he voiced the Japanese line himself.

The character W.D. Gaster shares some visual similarities with the character Uboa from Yume Nikki, another RPG-based title. Their appearance is also similar to Dr. Andonuts from Undertale creator Toby Fox's Halloween Hack for EarthBound.

While meeting Sans for the first time, Spoiler:Sans will ask the protagonist to turn around and shake his hand, which has a whoopie cushion hidden in it. However, if you immediately exit the game without saving and then meet him again, he will ask you if you've heard his joke before, since the main protagonist turns around before being asked.

Spoiler:After beating the game by only killing Toriel, Asgore, Papyrus, Mettaton, and Undyne, Sans will call the player after the Omega Flowey fight to tell them that the annoying dog became president.

The track "Waterfall" almost exactly matches the melody of "Spear of Justice", but at about a third of the speed.

By speeding up the song "The Ruins", it is almost identical to the songs "NGAHHH!!" and "Spear of Justice".

The opera scene with Mettaton is likely a parody of "Aria di Mezzo Carattere" from Final Fantasy VI.

The song "But Nobody Came" sped up 1600% sounds similar to another Undertale song, "Your Best Friend".

In the game's demo as well as full version prior to patch v1.001, an unused song by the name of "grandpasemi.ogg" could be found in the game's files. This was likely intended for a cut character that could have been related to Sans, Papyrus, and possibly even W.D. Gaster, due to the name Semi being based on Semi Serif fonts. It is a short, slowed-down, and re-instrumentated version of the song Metal Crusher, which is used as Mettaton's first battle theme.

This same song is now used as of patch v1.001 when you deny feeding Temmie any Tem Flakes. It plays while Temmie makes a unique facial expression, with a text graphic appearing beside her that says "can't blame a BARK for tryin'...", immediately followed by Temmie trying to attack you with her extendable arm.

The game contains an unused battle with "RG 03" and "RG 04", which appears to be mostly complete with the exception of using placeholder graphics.

The song "Death By Glamour" is a remix of the songs "Core", "Metal Crusher", and "It's Showtime".

If you attack the dummy inside of the ruins, but attack and miss it nine times in a row, a text will pop up saying "Dummy tires of your aimless shenanigans." This will automatically end the battle, earning you 0 gold and 0 XP.

The song "Dating Start!" sounds almost exactly like the song "Turnabout Sisters" from Pheonix Wright: Ace Attorney

The song "Pathitic House" sped up sounds similiar to the song "Spider Dance".

During then credits after the Spoiler:pacifist run, if you dodge everything, yellow text will appear saying "Incredible work! Somewhere, felt a door opening...!". This unlocks a door in Snowdin Forest.

There are unused graphics of a robot NPC in idle and talking animation stored in the game's memory.

According to developer Toby Fox, the song "Redline Day" helped inspire him to make changes to the Undertale OST.