Oxidative stress is the main inducer of β-cell damage, which underlies the pathogenesis of diabetes. Evidence suggests that glycine, a recognized antioxidant, may improve β-cell function; however, its mechanism in protecting diabetic β-cells against oxidative stress has not been directly investigated. Using a streptozotocin-induced diabetic rat model and INS-1 pancreatic β-cells, we evaluated whether glycine can attenuate diabetic β-cell damage induced by oxidative stress. In diabetic rats, glycine stimulated insulin secretion; enhanced plasma glutathione (GSH), catalase and superoxide dismutase levels; reduced plasma 8-hydroxy-2 deoxyguanosine and islet p22phox levels; and improved islet β-cell mitochondrial degeneration and insulin granule degranulation. In INS-1 cells, glycine reduced the intracellular reactive oxygen species (ROS) concentration and inhibited apoptosis induced by high glucose or H2O2. Glycine transporter-1 inhibitor blocked the antioxidative effect of glycine by reducing the intracellular GSH content, and glycine receptor inhibitor reversed the glycine antioxidative effect by blocking p22phox. Collectively, our findings reveal a mechanism by which glycine protects diabetic β-cells against damage caused by oxidative stress by increasing glycine transporter-1-mediated synthesis of GSH and by reducing glycine receptor-mediated ROS production.