Regular exercise has an antidepressant effect in human subjects. Studies using animals have suggested that the antidepressant effect of exercise is attributable to an increase of brain 5-hydroxytryptamine (5-HT); however, the precise mechanism underlying the antidepressant action via exercise is unclear. In contrast, the effect of 5-HT on antidepressant activity has not been clarified, in part because the therapeutic response to antidepressant drugs has a time lag in spite of the rapid increase of brain 5-HT upon administration of these drugs. This study was designed to investigate the contribution of brain 5-HT to the antidepressant effect of exercise. Mice were fed a tryptophan-deficient diet and stressed using chronic unpredictable stress (CUS) for 4 weeks with or without the performance of either moderate or intense exercise on a treadmill 3 days per week. The findings demonstrated that the onset of depression-like behavior is attributable not to chronic reduction of 5-HT but to chronic stress. Regular exercise, whether moderate or intense, prevents depression-like behavior with an improvement of adult hippocampal cell proliferation and survival and without the recovery of 5-HT. Concomitantly, the mice that exercised showed increased hippocampal noradrenaline. Regular exercise prevents the impairment of not long-term memory but short-term memory in a 5-HT-reduced state. Together, these findings suggest that: (1) chronic reduction of brain 5-HT may not contribute to the onset of depression-like behavior; (2) regular exercise, whether moderate or intense, prevents the onset of chronic stress-induced depression-like behavior independent of brain 5-HT and dependent on brain adrenaline; and (3) regular exercise prevents chronic tryptophan reduction-induced impairment of not long-term but short-term memory.