Mach Rider takes place in the year 2112, and planet Earth has been invaded by evil forces known as Quadrunners. The player controls Mach Rider, who travels from sector to sector on a motorcycle, searching for survivors and destroying the enemies in his path.

The game's controls are somewhat more complex than other games at its time. The left and right directions on the Control Pad steer Mach Rider and the A button accelerates. The B button fires Mach Rider's machine gun which can be used to destroy enemies and obstacles on the road. The up and down buttons are used to change gears. Mach Rider's bike has four gears and travels at an immense speed in top gear.

In each round, points can be scored by destroying enemies and certain obstacles with the machine gun. The number of points scored for destroying enemies and obstacles are determined by the power of the enemy destroyed and the type of obstacle destroyed. If the player "BLOCKS" an enemy by knocking it into a hazard on the track, they obtain more points, and this also replenishes Mach Rider's bullets.

Fighting Course: The Fighting Course consists of the primary story sequence. The player controls Mach Rider as he travels across ten different sectors, and tries to avoid being destroyed by obstacles such as Oil Drums and his enemies, the Quadrunners. If he is destroyed, he will separate into fragments and then reform, as long as he has energy remaining. Much like other games of the time, such as Ice Climber and Balloon Fight, there is no ending sequence. Once the tenth sector is completed, the story starts anew, with a second quest of ten entirely new sectors. On each sector in Fighting mode, the player is given the choice to ride to the next sector on either Track A or Track B. The two tracks are different from one another and with each new sector there are new tracks.

Endurance Course: The player must race a certain number of kilometres within a time limit while enemies and obstacles get in the way and slow down the progress. Lives and energy are not a factor in Endurance, but being destroyed causes a loss of precious time.

Solo Course: This is the same as Endurance Course, but with no enemies.

Design Mode: This is where the player may design their own sector and then race on it using one any of the three other modes of play. However, like Excitebike, the original game required the Famicom Data Recorder in order to save the tracks, and the device was never released outside Japan. The 2007 Virtual Console version of the game allows the user to save the tracks onto the Wii's built-in memory.

Back in 1972, Mach Rider was originally released as a plastic race car or hot rod toy also by Nintendo that came with a ramp for jumping and a stick shift-like object with three different kinds of meters on it. When the car is placed inside of it, it can be charged up and let loose at high speed.

Later, Mach Rider was released as part of the Nintendo Vs. Series arcade variations. It was essentially a modified version of the Endurance Course from the original game. It included a brief scene that for the first time revealed clearly what Mach Rider looked like. After the player completes all ten courses, it is shown that Mach Rider is a brown-haired woman in a white bikini holding a knife.

While Mach Rider has not gained a true sequel, many elements from the game, such as the futuristic setting, incredible high speeds and aggressive racing have also been used in Nintendo's premiere racing series F-Zero, making it somewhat of a spiritual sequel to Mach Rider. Even the character Mach Rider himself bears some similarity to the hero of the F-Zero series, Captain Falcon.

In the Super Smash Bros. series there were some Mach Rider references. Starting with Super Smash Bros. Melee, Nintendo's crossover fighting game has a remix medley of the Mach Rider soundtrack, including the title screen music, track selection music, stage music and game over jingle. This music is used as alternate music on the F-Zero themed stage called Big Blue, further reinforcing the connection between the two series. There is a Trophy of Mach Rider that can be earned, which shows Mach Rider in great detail on his motorcycle. The Trophy entry, which gives additional information about the plot of the game reads:

After the destruction of his home town, Mach Rider jumped onto his machine-gun-equipped combat motorcycle and set off in search of a new home, destroying all the Quadrunners who got in his way. His motorcycle had four gears and endless ammunition. When hit, Mach Rider would break into fragments and then rejoin together.

In Super Smash Bros. Brawl, the Mach Rider became one of the game's many Stickers, while the remix medley from Super Smash Bros. Melee returned, only this time to the F-Zero-themed stage, "Port Town: Aero Dive", rather than Big Blue.

In WarioWare: Twisted!, one of the many microgames is based on Mach Rider.