We recently reported that brain α7 nicotinic receptor (α7 nAChR) stimulation and brain hydrogen sulfide (H₂S) inhibited the rat micturition. In this study, we examined whether brain H₂S is involved in the micturition inhibition induced by brain α7 nAChR stimulation in urethane-anesthetized (0.8 g/kg, ip) male Wistar rats. A catheter was inserted into the bladder to perform cystometry (12 ml/h saline infusion). We examined effects of intracerebroventricularly (icv) pretreated GYY4137 (GYY, H₂S donor, 1 or 3 nmol/rat) or AOAA (non-selective inhibitor of H₂S synthesis, 3 or 10 μg/rat) on PHA568487 (PHA, α7 nAChR agonist, 0.3 or 1 nmol/rat, icv)-induced prolongation of intercontraction intervals (ICI), an index of micturition frequency. PHA (0.3 nmol/rat) showed no significant effect on ICI, while under pretreatment with GYY, PHA significantly prolonged ICI even at a lower dose (0.3 nmol/rat). PHA (1 nmol/rat) induced ICI prolongation and the PHA-induced prolongation was significantly suppressed by AOAA. The AOAA-induced suppression of the PHA-induced ICI prolongation was cancelled by supplementation of brain H₂S via GYY. These data suggest that brain α7 nAChR stimulation inhibits the rat micturition via brain H₂S.