Insulin resistance, a hallmark of type 2 diabetes, is associated with oxidative stress. However, the role of reactive oxygen species or specific antioxidant enzymes in its development has not been tested under physiological conditions. The objective of our study was to investigate the impact of overexpression of glutathione peroxidase 1 (GPX1), an intracellular selenoprotein that reduces hydrogen peroxide (H(2)O(2)) in vivo, on glucose metabolism and insulin function. The GPX1-overexpressing (OE) and WT male mice (n = 80) were fed a selenium-adequate diet (0.4 mg/kg) from 8 to 24 weeks of age. Compared with the WT, the OE mice developed (P < 0.05) hyperglycemia (117 vs. 149 mg/dl), hyperinsulinemia (419 vs. 1,350 pg/ml), and elevated plasma leptin (5 vs. 16 ng/ml) at 24 weeks of age. Meanwhile, these mice were heavier (37 vs. 27 g, P < 0.001) and fatter (37% vs. 17% fat, P < 0.01) than the WT mice. At 30-60 min after an insulin challenge, the OE mice had 25% less (P < 0.05) of a decrease in blood glucose than the WT mice. Their insulin resistance was associated with a 30-70% reduction (P < 0.05) in the insulin-stimulated phosphorylations of insulin receptor (beta-subunit) in liver and Akt (Ser(473) and Thr(308)) in liver and soleus muscle. Here we report the development of insulin resistance in mammals with elevated expression of an antioxidant enzyme and suggest that increased GPX1 activity may interfere with insulin function by overquenching intracellular reactive oxygen species required for insulin sensitizing.