Peripheral hydrogen sulfide (H₂S) is reported as an endogenous relaxation factor in the bladder, while in the central nervous system, roles of H₂S in regulation of micturition is unclear. In this study, therefore, we examined effects of centrally administered GYY4137 (GYY, H₂S donor) or AOAA (H₂S synthesis inhibitor), on the micturition reflex in urethane-anesthetized (0.8 g/kg, ip) male Wistar rats. A catheter was inserted into the bladder to perform cystometrograms (CMG). CMG was started 2 h after the surgery and 1 h after the start, GYY (3 or 10 nmol/rat) or AOAA (30 or 100 μg/rat) was intracerebroventricularly (icv) administered. Effects of icv pretreated SR95531 (SR, GABA_A antagonist, 0.1 nmol/rat) or SCH50911 (SCH, GABA_B antagonist, 0.1 nmol/rat) on the GYY-induced responses were also examined. GYY dose-dependently prolonged intercontraction intervals (ICI), while AOAA dose-dependently shortened ICI. The AOAA-induced ICI shortening was reversed in the presence of GYY. Pretreatment with SR or SCH significantly attenuated the GYY-induced ICI prolongation. These results suggest that brain H₂S can inhibit the micturition reflex via brain GABA_A and GABA_B receptors in rats.