Objective The pathogenesis of visceral hypersensitivity, a characteristic pathophysiological feature of irritable bowel syndrome (IBS), remains unknown.Recent studies suggest a role for hydrogen sulfide (H2S) in pain signaling but this has not been well studied in animal models of visceral hyperalgesia.We therefore determined the role for the endogenous H2S producing enzyme cystathionine-β-synthetase (CBS) and/or cystathionine-γ-lyase (CSE) in a validated rat model of IBS-like chronic visceral hyperalgesia (CVH).Methods Adult (6-8 weeks) male Sprague Dawley rats were subjected to consecutive 9 d of a heterotypical intermittent stress (HIS) protocol comprised of 3 randomly arranged stressors: 45 min cold restraint stress (CRS, 4 ℃), 20 min forced swimming stress (FSS, water temperature 22 ℃), and 60 min water avoidance stress (WAS, room temperature 22 ℃).The perception of painful sensation induced by colorectal distension (CRD) in conscious rats was measured by assessing the abdominal withdrawal reflex (AWR) scores at 6, 24, 48 and 108 h after termination of last stressor.CSE inhibitor DL-propargylglycine (PPG) or CBS inhibitor amino-oxyacetate (AOAA) was given intraperitoneally.Results HIS significantly increased AWR scores responding to CRD starting from 6 h and lasting for 48 h after determination of last stressor.PPG or AOAA attenuated the AWR scores induced by HIS, in a dose-dependent manner.The smallest dose for PPG and AOAA to produce maximal inhibitory effect was 50 mg/kg and 8 mg/kg body weight, respectively.Neither PPG nor AOAA produced any effect on AWR scores in normal rats.Conclusion These results suggest that CBS/CSE-H2S signaling pathways may play an important role in stress-induced visceral hyperalgesia, thus identifying a potential therapeutic molecule for functional gastrointestinal disorders such as IBS.