Pancreatic β-cells exposed to hyperglycemia produce reactive oxygen species (ROS). Because β-cells are sensitive to oxidative stress, excessive ROS may cause dysfunction of β-cells. Here we demonstrate that mitochondrial ROS suppress glucose-induced insulin secretion (GIIS) from β-cells. Intracellular ROS increased 15min after exposure to high glucose and this effect was blunted by inhibitors of the mitochondrial function. GIIS was also suppressed by H₂O₂, a chemical substitute for ROS. Interestingly, the first-phase of GIIS could be suppressed by 50μM H₂O₂. H₂O₂ or high glucose suppressed the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, and inhibitors of the mitochondrial function abolished the latter effects. Our data suggested that high glucose induced mitochondrial ROS, which suppressed first-phase of GIIS, at least in part, through the suppression of GAPDH activity. We propose that mitochondrial overwork is a potential mechanism causing impaired first-phase of GIIS in the early stages of diabetes mellitus.