Oxidative stress can contribute to the multifactorial etiology of skeletal muscle insulin resistance. No investigation has directly assessed the effect of in vitro oxidative stress on insulin action in mammalian skeletal muscle. The purpose of the present study was to characterize the molecular actions of a low-grade oxidant stress (hydrogen peroxide) on insulin signaling and glucose transport in skeletal muscle of lean Zucker rats. Soleus strips were incubated in 8 mM glucose for 2 hr in the absence or presence of 100 mU/ml glucose oxidase, which produces hydrogen peroxide at ~90 µM. By itself, hydrogen peroxide significantly (p<0.05) activated glucose transport activity, net glycogen synthesis, and glycogen synthase activity and increased phosphorylation of insulin receptor (tyrosine), Akt (ser⁴⁷³), and GSK-3 (ser⁹). In contrast, this oxidant stress significantly inhibited the expected insulin-mediated enhancements in glucose transport, glycogen synthesis, and these signaling factors, and allowed GSK-3 to retain a more active form. In the presence of CT98014, a selective GSK-3 inhibitor, the ability of insulin to stimulate glucose transport and glycogen synthesis during exposure to this oxidant stress was enhanced by 20% and 39%, respectively, and insulin stimulation of the phosphorylation of IR, Akt, and GSK-3 were all significantly increased by 36-58%. These results indicate that an oxidant stress can directly and rapidly induce substantial insulin resistance of skeletal muscle insulin signaling, glucose transport, and glycogen synthesis. Moreover, a small but significant portion of this oxidative stress-induced insulin resistance is associated with a reduced insulin-mediated suppression of the active form of GSK-3.