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.

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.

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".

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.