Jetpack Joyride casts players in the role of one Barry Steakfries, a character who has appeared in a number of Halfbrick's previous mobile titles. For reasons that are never made entirely clear, Barry bursts through the wall of a secret laboratory, steals a jetpack that is actually a machine gun and then attempts to make his escape by continually running in the opposite direction to the wall he burst in through.

Along the way, he must avoid the lab's overzealous security system, which includes floating electric gates, missiles, laser beams and numerous other hazards. Controlling Barry is simple. Clicking and holding the mouse button causes him to fire his jetpack, causing him to rise. Releasing causes him to fall. As he progresses along his endless run, he will come across collectible vehicles, each of which respond slightly differently to a click.

"Mr. Cuddles" the dragon, for example, floats at the top of the screen by default but swoops down while the mouse button is held. The "Profit Bird" vehicle flaps its wings when the mouse button is clicked, requiring repeated clicking to rise. And the "Crazy Freaking Teleporter" zaps Barry to a new location on the screen when the button is clicked.