Hydrogen sulfide (H2S) plays important roles in the enteric system in the wall of the gastrointestinal tract. There have been no studies on whether H2S is endogenously generated in peripheral sympathetic ganglia and, if so, its effect on synaptic transmission. In this study, we examined the effect of H2S on cholinergic excitatory fast synaptic transmission in the mouse superior mesenteric ganglion (SMG). Our study revealed that NaHS and endogenously generated H2S selectively potentiated cholinergic fast EPSPs (F-EPSPs) evoked by splanchnic nerve stimulation but not F-EPSPs evoked by colonic nerve stimulation. The H2S-producing enzyme cystathionine-γ-lyase (CSE) was expressed in both neurons and glial cells. The CSE blocker PAG (dl-propargylglycine) significantly reduced the amplitude of F-EPSPs evoked by splanchnic nerve stimulation but not F-EPSPs evoked by colonic nerve stimulation. Inhibiting the breakdown of endogenously generated H2S with stigmatellin potentiated the amplitude of F-EPSPs evoked by splanchnic nerve stimulation but not F-EPSPs evoked by colonic nerve stimulation. Splanchnic F-EPSPs but not colonic F-EPSPs were reduced in CSE knock-out (KO) mice. Functional studies showed that NaHS enhanced the inhibitory effect of splanchnic nerve stimulation on colonic motility. Colonic motility in CSE-KO mice was significantly higher than colonic motility in wild-type mice. We conclude that endogenously generated H2S acted selectively on presynaptic terminals of splanchnic nerves to modulate fast cholinergic synaptic input and that this effect of H2S modulates CNS control of gastrointestinal motility. Our results show for the first time that the facilitatory effect of endogenous H2S in the mouse SMG is pathway specific.