The Monolith tool that's used to raise a creature to the next technological level is a nod to the monolith from the film "2001 A Space Odyssey". In the film, mankind's early ancestors were given intelligence from a mysterious black monolith from space.

When the player's creature evolves into the tribe stage, a cutscene will play in which the creature learns how to use a stick as a weapon, then, as a way of celebration, throws the stick into the air and it lands on the creature's head.

This cinematic sequence is a parody of the opening sequence from the Stanley Kubrick film 2001: A Space Odyssey, where an ape learns how to use a bone as a weapon. Additionally, the intro cutscene from Spore features a remix of 'Sunrise', which is the initial fanfare of the tone poem "Also Sprach Zarathustra".

When trading with any empire, the theme song from the 1983 game M.U.L.E., an influential multiplayer game published in North America by EA, can sometimes be heard in the background. A song also called "M.U.L.E." can be found in the song library for use in adventures.

On the main menu, if you drag the galaxy and spin it around long enough, four pictures of the game's developers will fly out of the galaxy. However, if you do this while pressing Ctrl+Shift, even more developer pictures will appear.

During the Tribal, Civilization, and Space stages of the game, the characters will have discussions with speech bubbles above their head. These speech bubbles will contain various objects, one of which being a Plumbob. This is a reference to The Sims series.

There is a planet that shows up in Spore called Tamaran. This is a reference to the home planet of the DC Comics character Starfire.

The achievement "My Precious" is a reference to the iconic phrase said often by Gollum from the Lord of the Rings series.

The "Oh The Humanity!" achievement is a reference to a phrase said during the broadcast of the Hindenburg disaster by American radio journalist Herbert Morrison.

There is a tool in Spore called the "Happiness Booster" and at its base are the letters: IPPETAD. These letters, as well as the tool itself, are a reference to the Family Guy/Star Wars crossover episode "Blue Harvest" where the character 'Darth' Al Harrington is selling Intergalactic Proton Powered Electrical Tentacle Advertising Droids. When initialized this spells IPPETAD.

There is a character that can be found at the center of the galaxy by the name of "Steve". He pilots a spaceship nearly identical to the one on the cover of SimCity 2000.

In the Vehicle Creator, there's a Body part named the Kessel Runner. This is a reference to the hyperspace route named the Kessel Run in the Star Wars series which Han Solo notably used to tout the abilities of the Millennium Falcon.

There is an achievement in the game called "Village Folks" accompanied with an image showing four creatures forming the letters Y M C A. The name is a reference to the band Village People and the image is referencing their hit song "YMCA".