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In Super Mario Bros., the player takes on the role of Mario, the protagonist of the series. Mario's younger brother, Luigi, is controlled by the second player in the game's multiplayer mode and assumes the same plot role and functionality as Mario. The objective is to race through the Mushroom Kingdom, survive the main antagonist Bowser's forces, and save Princess Toadstool.^[3]:7 The game is a side-scrolling platformer; the player moves from the left side of the screen to the right side in order to reach the flag pole at the end of each level.

The game world features coins scattered around for Mario to collect and special bricks marked with a question mark (?), which when hit from below by Mario may reveal more coins or a special item. Other "secret", often invisible, bricks may contain more coins or rare items. If the player gains a Super Mushroom, Mario grows to double his size and gains the ability to break bricks above him. If Mario gets hit in this mode, then instead of dying he turns back to regular Mario.^[3]:12 Players start with a certain number of lives and may gain additional lives by picking up green spotted orange 1-Up mushrooms hidden in bricks, or by collecting 100 coins, defeating several enemies in a row with a Koopa shell, or bouncing on enemies successively without touching the ground. Mario loses a life if he takes damage while small, falls in a bottomless pit, or runs out of time. The game ends when the player runs out of lives, although a button input can be used on the game over screen to continue from the first level of the world in which the player died.^[4]

Mario's primary attack is jumping on top of enemies, though many enemies have differing responses to this. For example, a Goomba will flatten and be defeated,^[3]:12 while a Koopa Troopa will temporarily retract into its shell, allowing Mario to use it as a projectile.^[3]:11 These shells may be deflected off a wall to destroy other enemies, though they can also bounce back against Mario, which will hurt or kill him.^[3]:19 Other enemies, such as underwater foes and enemies with spiked tops, cannot be jumped on and damage the player instead. Mario can also defeat enemies above him by jumping to hit the brick that the enemy is standing on. Mario may also acquire the Fire Flower from certain "?" blocks that when picked up changes the color of Super Mario's outfit and allows him to throw fireballs. However, certain enemies such as Buzzy Beetles are immune to fireballs. A less common item is the Starman, which often appears when Mario hits certain concealed or otherwise invisible blocks. This item makes Mario temporarily invincible to most hazards and capable of defeating enemies on contact.^[3]:10

The game consists of eight worlds with four sub-levels called "stages" in each world."^[5][3]:7 The final stage of each world takes place in a castle where Bowser is fought above a suspension bridge; the first seven of these Bowsers are "false Bowsers" whom are actually minions disguised as him, whilst the real Bowser is found in the 8th world. Bowser and his decoys are defeated by jumping over them and reaching the axe on the end of the bridge, although they can also be defeated using a Fire Flower. The game also includes some stages taking place underwater, which contain different enemies. In addition, there are bonuses and secret areas in the game. Most secret areas contain more coins for Mario to collect, but some contain "warp pipes" that allow Mario to advance directly to later worlds in the game without completing the intervening stages. After completing the game once, the player is rewarded with the ability to replay the game with changes made to increase its difficulty, such as all Goombas in the game being replaced with Buzzy Beetles.