Super Mario Run plays as a side-scrolling, auto-runner platform game. The plot begins with Mario accepting Princess Peach's invitation to her castle, only to witness Bowser kidnapping the Princess and destroying the Mushroom Kingdom, tasking Mario to undo his actions. Mario automatically runs from left to right and jumps on his own to clear small gaps or obstacles.

The player can control Mario by tapping the touch screen to make him jump over larger obstacles; the longer the screen is touched, the higher he jumps. Like other Super Mario games, the player must maneuver Mario over gaps, onto enemies, and into coins to collect them. The player's end goal is to not only steer Mario safely across the level and reach the flagpole before time is exhausted but also accumulate as many coins as possible.

In addition to controlling when and how high he jumps, a hidden ability exists where the player swipes and holds the screen in the direction opposite of Mario, stalling his jumps during his descent and moving him slightly in that direction. Standing on pause blocks pauses the timer and Mario's running, and standing on blocks marked with directional arrows changes the direction of his jump. Bubbles are collectible items used to send him backward, letting the player repeat sections of the level.

If a bubble is available, it is automatically used to rescue Mario from impending death. Otherwise, the player can only restart the level. Replayability comes from collecting all the normal coins and finding special coins in each level. First, the player must find five pink coins, collecting those unlocks five purple coins and finally collecting those unlocks five black coins, therefore it takes at least three playthroughs to collect everything in a given level.

In addition to the main game, there is the "Toad Rally" game mode where players can challenge "ghost" versions of other players' prerecorded playthroughs of levels. Access to Toad Rally requires the player to use a Rally Ticket, which can be earned either through completing levels in the main game mode, trading My Nintendo coins, or other in-game tasks.

Playing either game mode earns the player coins that can be used to buy items for creating and customizing their own "Mushroom Kingdom" in a third gameplay mode comparable to FarmVille. The player can gain or lose Toads that populate their Mushroom Kingdom in Toad Rallies. The player can unlock other characters to play as through these game modes, such as Luigi, Princess Peach, Toad, Toadette, and Yoshi.

These characters have slightly different gameplay attributes; using the different characters is not required to complete any level, but they give players the tools for completing levels with different approaches. Since launch, Nintendo has released updates introducing features and other changes. For example, a "Friendly Run" version of Toad Rally does not require any Rally Tickets, but can only be played up to five times per day, and will not earn the player coins for winning.

An easy difficulty mode assists players having difficulty in beating levels in World Tour by granting them unlimited bubbles and removing the time limit. However, coins collected will not count while playing in that mode. Another update raised the maximum cap number of Toads from 9,999 to 99,999, and added support for Game Center and Google Play Games achievements.

A later update added nine new levels, courses, Princess Daisy as a playable character, and buildings, all of which can be unlocked by completing various goals and challenges. The levels are themed as "a forest, a ship packed with coins, and a whole airship armada", and a new game mode, titled "Remix 10", has players run through ten very short levels in a random order, with each level only taking "a few seconds to complete."