T he enhanced oxidative stress associated with type 2 diabetes mellitus contributes to disease pathogenesis. We previously identified plasma membrane–associated ATP-sensitive K⁺ (K_ATP) channels of pancreatic β cells as targets for oxidants. Here, we examined the effects of genetic and pharmacologic ablation of K_ATP channels on loss of mouse β cell function and viability following oxidative stress. Using mice lacking the sulfonylurea receptor type 1 (Sur1) subunit of K_ATP channels, we found that, compared with insulin secretion by WT islets, insulin secretion by Sur1^–/– islets was less susceptible to oxidative stress induced by the oxidant H₂O₂. This was likely, at least in part, a result of the reduced ability of H₂O₂ to hyperpolarize plasma membrane potential and reduce cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) in the Sur1^–/– β cells. Remarkably, Sur1^–/– β cells were less prone to apoptosis induced by H₂O₂ or an NO donor than WT β cells, despite an enhanced basal rate of apoptosis. This protective effect was attributed to upregulation of the antioxidant enzymes SOD, glutathione peroxidase, and catalase. Upregulation of antioxidant enzymes and reduced sensitivity of Sur1^–/– cells to H₂O₂-induced apoptosis were mimicked by treatment with the sulfonylureas tolbutamide and gliclazide. Enzyme upregulation and protection against oxidant-induced apoptosis were abrogated by agents lowering [Ca²⁺]_c. Sur1^–/– mice were less susceptible than WT mice to streptozotocin-induced β cell destruction and subsequent hyperglycemia and death, which suggests that loss of K_ATP channel activity may protect against streptozotocin-induced diabetes in vivo.