There is a significant interest in uncovering the mechanisms by which exercise improves cognition, memory, and mood, and reduces the risk for neurodegenerative disease. Here, we use light sheet 3D imaging in combination with c-Fos immunohistochemistry to create an atlas of exercise-induced brain activity in mice. Our dataset reveals 255 brain regions activated by acute exercise in mice, the majority of which have not previously been linked to exercise. We find 32 brain regions uniquely regulated by wheel running and 83 brain regions uniquely regulated by treadmill running. Yet, in contrast to voluntary wheel running, forced treadmill running triggers activity in brain regions associated with stress and pain, questioning the translational validity of forced treadmill running. In sum, we provide a comprehensive resource to support and guide future work focused on dissecting the neurobiological response to exercise.