Physical exercise can improve brain function; however, the effects of exercise cessation are largely unknown. This study examined the time-course profile of hippocampal neurogenesis following exercise cessation. Male C57BL/6 mice were randomly assigned to either a control (Con) or an exercise cessation (ExC) group. ExC mice were reared in a cage with a running wheel for 8 weeks and subsequently placed in a standard cage to cease the exercise. Exercise significantly increased the density of doublecortin (DCX)-positive immature neurons in the dentate gyrus (at week 0). Following exercise cessation, the density of DCX⁺ neurons gradually decreased and was significantly lower than that in Con at 5 and 8 weeks after cessation, indicating that exercise cessation leads to a negative rebound in hippocampal neurogenesis. Immunohistochemistry analysis suggested the negative rebound in neurogenesis to be caused by diminished cell survival, not by suppression of cell proliferation and neural maturation. Neither elevated expression of FosB, a transcription factor involved in neurogenesis regulation, nor increased plasma corticosterone were involved in the negative neurogenesis rebound. Importantly, exercise cessation suppressed the ambulatory activity, and a significant correlation between change in activity and DCX⁺ neuron density suggested that the decrease in activity is involved in the impairment of neurogenesis. Forced treadmill running following exercise cessation failed to prevent the negative neurogenesis rebound. This study indicates that cessation of exercise or a decrease in physical activity is associated with an increased risk of impaired hippocampal function, which might increase vulnerability to stress-induced mood disorders.