Transcription factor Nrf2 (NF-E2–related factor 2) regulates wide-ranging cytoprotective genes in response to environmental stress. Keap1 (Kelch-like ECH–associated protein 1) is an adaptor protein for Cullin3-based ubiquitin E3 ligase and negatively regulates Nrf2. The Keap1-Nrf2 system plays important roles in the oxidative stress response and metabolism. However, the roles Nrf2 plays in prevention of pancreatic β-cell damage remain elusive. To demonstrate the roles of Nrf2 in pancreatic β-cells, we used four genetically engineered mouse models: 1) β-cell–specific Keap1-conditional knockout mice, 2) β-cell–specific Nos2 transgenic mice, 3) conventional Nrf2-heterozygous knockout mice, and 4) β-cell–specific Nrf2-conditional knockout mice. We found that Nrf2 induction suppressed the oxidative DNA-adduct formation in pancreatic islets of iNOS-Tg mice and strongly restored insulin secretion from pancreatic β-cells in the context of reactive species (RS) damage. Consistently, Nrf2 suppressed accumulation of intracellular RS in isolated islets and pancreatic β-cell lines and also decreased nitrotyrosine levels. Nrf2 induced glutathione-related genes and reduced pancreatic β-cell apoptosis mediated by nitric oxide. In contrast, Nrf2 depletion in Nrf2-heterozygous knockout and β-cell–specific Nrf2-conditional knockout mice strongly aggravated pancreatic β-cell damage. These results demonstrate that Nrf2 induction prevents RS damage in pancreatic β-cells and that the Keap1-Nrf2 system is the crucial defense pathway for the physiological and pathological protection of pancreatic β-cells.