We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle, by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum longus (EDL) muscles to hypoxanthine/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine if the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of hydrogen peroxide (H₂O₂), EDL muscles from rats were preincubated with the H₂O₂ scavenger catalase or the superoxide scavenger superoxide dismutase (SOD) prior to incubation with Hx/XO. Catalase treatment but not SOD completely inhibited the increase in Hx/XO-stimulated 2-DG uptake, suggesting that H₂O₂ is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct H₂O₂ also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 µmol/l H₂O₂. H₂O₂-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin, but not the nitric oxide inhibitor, N^G-monomethyl-L-arginine. H₂O₂ stimulated the phosphorylation ofAkt Ser⁴⁷³ (7-fold) and Thr³⁰⁸ (2-fold) in isolated EDL muscles. H₂O₂ at 600 µmol/l had no effect on ATP concentrations and did not increase the activities of either the 1 or 2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI3K-dependent mechanism.