This study aimed to investigate the underlying mechanism of protease-activated receptor-2 (PAR-2) agonist-induced visceral hyperalgesia. Male Sprague-Dawley rat pups were submitted to colonic injection of PAR-2 agonist for 6 consecutive days. The visceral sensitivity to colorectal distention was evaluated by electromyography. The enterochromaffin (EC) cell number, 5-HT content and tryrptophan hydroxylase (TPH) protein expression were detected with immunohistochemistry, fluorescent measurement and Western blot analysis. PAR-2 agonist induced a significant increase of visceral nociceptive response to colorectal distention and a series of neurochemical changes in rat colon, including proliferation of EC cells, increased 5-HT content and enhanced TPH expression. Expression of PAR-2 in EC cells was reported for the first time. Further, selective 5-HT(3) receptor antagonist alosteron significantly inhibited PAR-2-induced visceral hyperalgesia. The enhanced 5-HT signaling is likely responsible for the visceral hyperalgesia induced by PAR-2 agonist. Interruption of this pathway is a possible target for the treatment of visceral hyperalgesia in gastrointestinal diseases.