Uncoupling protein (UCP) 2 is a widely expressed mitochondrial protein whose precise function is still unclear but has been linked to mitochondria-derived reactive oxygen species production. Thus, the chronic absence of UCP2 has the potential to promote persistent reactive oxygen species accumulation and an oxidative stress response. Here, we show that Ucp2-/- mice on three highly congenic (N >10) strain backgrounds (C57BL/6J, A/J, 129/SvImJ), including two independently generated sources of Ucp2-null animals, all exhibit increased oxidative stress. Ucp2-null animals exhibit a decreased ratio of reduced glutathione to its oxidized form in blood and tissues that normally express UCP2, including pancreatic islets. Islets from Ucp2-/- mice exhibit elevated levels of numerous antioxidant enzymes, increased nitrotyrosine and F4/80 staining, but no change in insulin content. Contrary to results in Ucp2-/- mice of mixed 129/B6 strain background, glucose-stimulated insulin secretion in Ucp2-/- islets of each congenic strain was significantly decreased. These data show that the chronic absence of UCP2 causes oxidative stress, including in islets, and is accompanied by impaired glucose-stimulated insulin secretion.