While serotonin (5-HT) may impair learning and memory, exercise has been reported to improve them. Whether chronic exercise can facilitate fear memory via regulating the serotonin system is unknown. We examined the effects of 4-week treadmill exercise training on levels of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), the protein expression of its receptor 5-HT(1A) and transporter in the amygdala, hippocampus and prefrontal cortex of male Sprague-Dawley rats. Our results demonstrated that treadmill exercise (1) improved the passive avoidance learning performance; (2) decreased the 5-HT level in the hippocampus; (3) decreased the expression of 5-HT(1A) receptor in the amygdala without altering the transporter expression. Moreover, pretreatment with 0.1 mg/kg 8-hydroxy-di-n-propylamino tetralin, a selective 5-HT(1A) receptor agonist, impaired the passive avoidance performance and completely abolished the exercise-enhanced fear memory. Our results suggest that down-regulation of the 5-HT system in the limbic system, i.e., the reduction of the hippocampus 5-HT content and the amygdala 5-HT(1A) receptor expression, may be involved in the exercise-enhanced fear memory.