Background NO and H2S are gaseous signaling molecules that modulate smooth muscle motility. NO has been proved to be an important non-adrenergic non-cholinergic (NANC) inhibitory neurotransmitter which is released in response to nerve stimulation and relaxes smooth muscles. And our previous studies showed smooth muscle tonic contraction to be increased significantly by low-concentration NaHS in the gastric antrum. But few studies have been performed in the GI tract to demonstrate the interaction between NO and H2S. We aimed to identify expressions of enzymes that catalyze H2S and NO generation in mouse gastric smooth muscle, and determine relationships between endogenous H2S and NO in regulating smooth muscle motility. Methods Adult male ICR mice were used in the present study. Western blotting and immunohistochemistry methods were used to track expressions of neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in gastric smooth muscle. Smooth muscle motility was recorded by isometric force transducers; cGMP production was measured by a specific radioimmunoassay. Key Results CBS, CSE, eNOS, and nNOS were all expressed in mice gastric antrial smooth muscle tissues, but only CBS and eNOS were detected in gastric antrial smooth muscle cells. Aminooxyacetic acid (AOAA), a blocker of CBS, significantly inhibited smooth muscle contractions in the gastric antrum, which was significantly recovered by NaHS, while DL-propargylglycine (PAG), a blocker of CSE, had no significant effect; Nω-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of NOS, enhanced contractions. NaHS at low concentrations increased basal tension but decreased it at high concentrations; SNP significantly inhibited the contractions, which could be significantly recovered by NaHS both in the absence and presence of CuSO4. 1H-[1, 2, 4]Oxadiazolo[4, 3-α]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, did not block NaHS-induced excitatory effect; 3-Isobutyl-1-methylxanthine (IBMX), a non-selective phosphodiesterase inhibitor, partially blocked this effect; cGMP production in smooth muscle was significantly increased by SNP but not affected by NaHS. Conclusion & inferences Endogenous H2S and NO appear to play opposite roles in regulating gastric motility; their effects may be via separate signal pathways. Intracellular H2S/NO levels may be maintained in a state of balance to warrant normal smooth muscle motility. Keywords: endogenous H2S, endogenous NO, gastric antrial smooth muscle, motility