We have reported protective effect of NaHS [hydrogen sulfide (H₂S) donor] on cyclophosphamide (CYP)-induced rat bladder dysfunction by improving CYP-induced shortening of intercontraction intervals (ICI) and increases in non-voiding contractions (NVCs). In this study, we examined mechanisms of the protective effect. Nine-week-old male Wistar rats were pretreated with NaHS (10 μmol/kg, ip) or saline once daily for 7 days. CYP (150 mg/kg, ip) or saline had been injected 2 days before urodynamic experiments, and after the experiments, bladder tissues were collected to perform HE staining. In some rats, vehicle or capsaicin (CAP, 125 mg/kg, sc), which can desensitize CAP-sensitive afferent nerves, was pretreated 4 days before urodynamic experiments. In bladder tissues, CYP increased neutrophile infiltration, bleeding, and edema, but NaHS partially improved only the edema. CAP prolonged ICI and reduced NVCs in CYP-treated rats. NaHS-induced improvement of CYP-induced ICI shortening and NVC increasing was not detected in CAP-treated rats. These data suggest that NaHS showed protective effect on bladder dysfunction in CYP-treated rats via suppression of CAP-sensitive bladder afferent nerves but not of bladder inflammation.