One of the unique features of β-cells is their relatively low expression of many antioxidant enzymes. This could render β-cells susceptible to oxidative damage but may also provide a system that is sensitive to reactive oxygen species as signals. In isolated mouse islets and INS-1(832/13) cells, glucose increases intracellular accumulation of H₂O₂. In both models, insulin secretion could be stimulated by provision of either exogenous H₂O₂ or diethyl maleate, which raises intracellular H₂O₂ levels. Provision of exogenous H₂O₂ scavengers, including cell permeable catalase and N-acetyl-l-cysteine, inhibited glucose-stimulated H₂O₂ accumulation and insulin secretion (GSIS). In contrast, cell permeable superoxide dismutase, which metabolizes superoxide into H₂O₂, had no effect on GSIS. Because oxidative stress is an important risk factor for β-cell dysfunction in diabetes, the relationship between glucose-induced H₂O₂ generation and GSIS was investigated under various oxidative stress conditions. Acute exposure of isolated mouse islets or INS-1(832/13) cells to oxidative stressors, including arsenite, 4-hydroxynonenal, and methylglyoxal, led to decreased GSIS. This impaired GSIS was associated with increases in a battery of endogenous antioxidant enzymes. Taken together, these findings suggest that H₂O₂ derived from glucose metabolism is one of the metabolic signals for insulin secretion, whereas oxidative stress may disturb its signaling function.