Players control one of eight Mario franchise characters, who race around 16 tracks (four in each of four cups) with items that can either harm opponents or aid the user.

During a race, the players can obtain random items from special boxes placed in different areas of the track that are used to impede the opposition and gain the advantage.

On Yoshi Valley, the second race of the Special Cup, the rectangular progress bar does not identify characters due to the vagueness of the course's paths.

[23][24][25] Miyamoto stated that the game was 95% complete, but Nintendo chose not to display a playable version due to the difficult logistics of demonstrating the multiplayer features.

[24] The prototype featured the Feather item from Super Mario Kart and a Magikoopa as one of the eight playable characters, who was replaced with Donkey Kong in the final game.

[29] Halfway into the game's production, the developers suffered a hard disk crash, causing the original character models to become lost.

[30] The technique of rubber band AI prevents all the racers from easily separating, and the Spiny Shell item, which targets and attacks the player in first place, was added in order to keep each race competitive and balanced.

[37][52] Carine Barrel from the French Officiel Nintendo Magazine enjoyed the game's colourful and fluid visuals, adding that its overall presentation likened a "magical" experience.

[61] Detractors of the graphics felt they lacked detail (Tom Gulse from Computer and Video Games and Peer Schneider from IGN),[46][11] weren't better enough than the previous 16-bit entry (AllGame's Scott McCall and Neil West from Next Generation),[45][57] and failed to fully benefit from the N64's power (Francois Caron of Jeuxvideo.com).

[55] Morley disliked Mario Kart 64's wide, motorway-like track design by saying that it did not provide an "adrenaline filled" experience which players might have hoped for.

[60] Critics also found fault in the game's use of rubberband difficulty balancing, recognising that it gave the enemy AI an unfair advantage.

[54][63] Hyper's David Wildgoose and Jonathan highlighted the flexible turning control with the multiple-angled joystick, calling it "perfect" and true to real-life karts.

[55] Wildgoose reported having many unexpected moments while playing the game due to its "ingeniously fiendish AI" and the boxes containing different power-ups each time they're collected.

[53] Reviewers, even those lukewarm towards the graphics, positively noted touches such as the 180-degree turns in Bowser's Castle, the train tracks on Kalimari Desert, the trucks in Toad's Turnpike, the cows in Moo Moo Farm, Peach's castle on Royal Raceway, and the sliding penguins in Sherbet Land as highlights, as well as smoke puffs coming out of the kart.

[68] In addition to time trial world records, which have been documented practically since the beginning and still persists to this day,[69] Mario Kart 64 has a long and rich speedrunning history, often being the subject of speedrunning documentarian Summoning Salt through both methods of competitively playing the game in single-player mode, whose videos on Mario Kart 64 alone have racked up more than 26 million views as of October 2023.