Prior to the release of the official Japanese version, Japanese audiences, who previously experienced the game through various fan translations, frequently debated about how Sans would refer to himself in dialogue. In the Japanese language, there are multiple first-person singular pronouns, each with their own distinct connotations. Among Japanese fans, the common consensus was that Sans would either refer to himself with 僕 ("boku") or 俺 ("ore"). While both pronouns are masculine, the former has an informal and "boyish" connotation, while the latter typically indicates brashness.

Once the Japanese localization came out, audiences were surprised to discover that Sans instead refers to himself with オイラ ("oira"), a relatively archaic pronoun which is commonly associated with hillbillies (though milder in connotation than おら, "ora"), only using "ore" during his sterner moments. The atypical choice of pronoun resulted in #オイラショック ("#OiraShock") becoming a trending topic on Twitter in Japan, with many fanartists in the country responding by making tongue-in-cheek drawings riffing on it.

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 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.

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.

When an enemy without a death sprite dies, it will use Aaron's death sprite as place-holder for any enemy without one.

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.