Patients with type 2 diabetes lose β cells, but the underlying mechanisms are incompletely understood. Glucose-6-phosphate dehydrogenase (G6PD) is the principal source of the major intracellular reductant, NADPH, which is required by many enzymes, including enzymes of the antioxidant pathway. Previous work from our laboratory has shown that high glucose impairs G6PD activity in endothelial and kidney cells, which leads to decreased cell survival. Pancreatic β cells are highly sensitive to increased ROS. This study aimed to determine whether G6PD and NADPH play central roles in β-cell survival. Human and mouse islets, MIN6 cell line, and G6PD deficient mice were studied. High glucose inhibited G6PD expression and activity. Inhibition of G6PD with siRNA led to increased ROS and apoptosis, decreased proliferation, and impaired insulin secretion. High glucose decreased insulin secretion, which was improved by overexpressing G6PD. G6PD-deficient mice had smaller islets and impaired glucose tolerance compared with control mice, which suggests that G6PD deficiency per se leads to β-cell dysfunction and death. G6PD plays an important role in β-cell function and survival. High-glucose-mediated decrease in G6PD activity may provide a mechanistic explanation for the gradual loss of β cells in patients with diabetes.—Zhang, Z., Liew, CW, Handy, DE, Zhang, Y., Leopold, JA, Hu, J., Guo, L., Kulkarni, RN, Loscalzo, J., Stanton, RC High glucose inhibits glucose-6-phosphate dehydrogenase, leading to increased oxidative stress and β-cell apoptosis.