Effect of caffeine on the expression of tryptophan hydroxylase (TPH), rate limiting enzyme of serotonin synthesis, in dorsal and median raphe was investigated via immunohistochemistry. In exercise groups, Sprague-Dawley rats were put on treadmill running for 30 min per day for 6 consecutive days. On the seventh day, animals of control-with-caffeine group were injected subcutaneously with 4 mg/kg caffeine, while control-without-caffeine group were injected with 0.9% NaCl, sacrificed 2 h later. Exercise-with-caffeine group and exercise-without-caffeine group were injected with caffeine and NaCl, respectively; all-out time was determined 1 h after injection, and then sacrificed. Caffeine increased all-out time in exercised rats, and inhibited the exercise-induced elevation in TPH expression. The suppressive effect of caffeine on TPH expression in exercised rats can be suggested as one possible ergogenic mechanism of caffeine.