Beta cell dysfunction and death play a key role during the development of type 2 diabetes mellitus and are partly mediated by oxidative stress. We have reported previously that K_ATP channel knockout (SUR1-KO) decreases the susceptibility of pancreatic beta cells to ROS-induced cell damage. In glucose-stimulated wildtype (WT) islets we observed that insulin secretion was impaired by H₂O₂ concentrations down to 10 mM, whereas hormone release from SUR1-KO mice was not affected at low H₂O₂ concentrations. In WT beta cells this impact on cell function was coupled to a decrease in cell viability under conditions of oxidative stress. Importantly, the protective effect of genetic K_ATP channel ablation on cell viability could also be mimicked by preincubating WT cells with a low concentration of the sulfonylurea tolbutamide (25 mM). Under these conditions, similar to the situation in SUR1-KO beta cells, basal apoptosis was increased but beta cells were protected against H₂O₂ (10 mM). In order to elucidate the underlying protective mechanism, we determined the activity of the three most important antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (Cat). Islets from SUR1-KO mice showed an increase in SOD activity compared to WT islets (364 vs 243 U/mg protein, n=4, P<0.05). Furthermore, GPx and Cat activities were approximately doubled in SUR1-KO vs WT islets (0.370.02 vs 0.200.02 U/mg protein, n=5, P<0.05; 251 vs 141 mU/mg protein, n=3, P<0.001). Incubating WT islets with 100 mM tolbutamide for 4 hours resulted in an augmentation of SOD activity (323 vs 242 U/mg protein, n=6, P<0.05), too. Consequently, modulation of K_ATP channel activity preserved cell function and viability under conditions of oxidative stress. This is due to an upregulation of antioxidant enzymes. Regarding the fact that pancreatic beta cells are poorly equipped with antioxidant defence mechanisms, inhibiting K_ATP channels may be a promising approach to counteract ROS-induced beta cell damage during the pathogenesis of diabetes mellitus and islet transplantation.