Hydrogen sulfide (H2S) was historically recognized as a toxic gas generated by natural resources. However, its enzymatic production from L-cysteine has recently been demonstrated in mammals. Cystathionine β-synthase and cystathionine γ-lyase, both of which can produce H2S, were expressed in mouse pancreatic islet cells and the β-cell line, MIN6. L-Cysteine and the H2S donor NaHS inhibited glucose-induced insulin release from islets and MIN6 cells. These inhibitory effects were reproduced when insulin release was stimulated by α-ketoisocaproate, tolbutamide, or high K+. L-Cysteine and NaHS inhibited glucose-potentiated insulin release in the copresence of diazoxide and high K+. Real-time imaging of intracellular Ca2+ concentration ([Ca2+]i) demonstrated that both L-cysteine and NaHS reversibly suppressed glucose-induced [Ca2+]i oscillation in a single β-cell without obvious changes in the mean value. These substances inhibited Ca2+- or guanosine 5′-0-3-thiotriphosphate–induced insulin release from islets permeabilized with streptolysin-O. L-Cysteine and NaHS reduced ATP production and attenuated glucose-induced hyperpolarization of the mitochondrial membrane potential. Finally, L-cysteine increased H2S content in MIN6 cells. We suggest here that L-cysteine inhibits insulin release via multiple actions on the insulin secretory process through H2S production. Because the activities of H2S-producing enzymes and the tissue H2S contents are known to increase under diabetic conditions, the inhibition may participate in the deterioration of insulin release in this disease.