Uncoupling of oxidative phosphorylation represents a potential target for the treatment of hyperglycemia and insulin resistance in obesity and type 2 diabetes. AIMS. The current study investigated whether the expression of uncoupling protein 1 in skeletal muscles of transgenic mice (mUCP1 TG) modulates insulin action in major insulin target tissues in vivo. METHODS. Euglycemic-hyperinsulinemic clamps (17 pM/kg lean body mass/min) were performed in nine months old, hemizygous, male mUCP1 TG mice and WT littermates matched for body composition. RESULTS. mUCP1 TG mice exhibited fasting hypoglycemia and hypoinsulinemia compared to WT mice, whereas fasting hepatic glucose production rates were comparable in both genotypes. mUCP1 TG mice were markedly more sensitive to insulin action compared to WT mice and displayed three-fold higher glucose infusion rates, enhanced skeletal muscle and white adipose tissue glucose uptake, and whole body glycolysis rates. In the absence of alterations in plasma adiponectin concentrations, acceleration of insulin-stimulated glucose turnover in skeletal muscle of mUCP1 TG was accompanied by increased pAkt/Akt and pAMPK/AMPK ratios compared with WT mice. CONCLUSION. UCP1-mediated uncoupling of oxidative phosphorylation in skeletal muscle was paralleled by AMPK activation, and thereby stimulated insulin-mediated glucose uptake in skeletal muscle.