The player starts with the basic revolver, but they may upgrade by taking weapons from red chests and collect more ammunition from yellow chests. Upon reaching level 10, the player can choose a character-specific 'ultra-mutation'. The game has a leveling system, where the player gains experience in the form of radiation pellets, or 'rads', dropped by enemies, that allow them to get different mutations and choose what would benefit the character the most out of a selection of four, randomly selected mutations. Except for special weapons acquired in a hidden level and 'crowns' retained until after defeating the final boss, anything acquired in one playthrough does not carry over to the next. The user may continue looping indefinitely until death. After this, the player may choose to loop to the beginning of the game again with a greatly increased difficulty. The player progresses through the linear level structure until level 7-3, where the final boss, the Nuclear Throne, has to be defeated. Secondary sprites or skins can be acquired for the characters played if the player completes special or secret tasks. These individual abilities allow for different play-styles, improving the variety and replay value of the game. Each character has an ability unique to them, adding an additional element to the gameplay. Two additional secret characters can only be played under special circumstances during play.

The player controls one of a total of 12 characters, 10 of which must be unlocked through play.

There are also daily and weekly challenge modes, allowing the player to compete against others via the Steam platform for the best score (determined by the number of kills in the playthrough), on the same set of randomly generated levels. The game consists of two main game modes: single-player, and a local cooperative gameplay mode. Nuclear Throne is a top-down shooter roguelike game with bullet hell elements.

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