Hydrogen sulfide (H₂S), the third gasotransmitter, is endogenously generated by certain H₂S synthesizing enzymes, including cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) from L-cysteine in the mammalian body. Several studies have shown that endogenous and exogenous H₂S affects the proliferation of cancer cells, although the effects of H₂S appear to vary with cell type, being either promotive or suppressive. In the present study, we determined whether endogenously formed H₂S regulates proliferation in human gastric cancer AGS cells. CSE, but not CBS, was expressed in AGS cells. CSE inhibitors, DL-propargylglycine (PPG) and β-cyano-L-alanine (BCA), significantly suppressed the proliferation of AGS cells in a concentration-dependent manner. CSE inhibitors did not increase lactate dehydrogenase (LDH) release in the same concentration range. The inhibitory effects of PPG and BCA on cell proliferation were reversed by repetitive application of NaHS, a donor of H₂S. Interestingly, nuclear condensation and fragmentation were detected in AGS cells treated with PPG or BCA. These results suggest that endogenous H₂S produced by CSE may contribute to the proliferation of gastric cancer AGS cells, most probably through anti-apoptotic actions.