Increased cellular exposure to oxidants may contribute to the development of insulin resistance and type 2 diabetes. Skeletal muscle is the primary site of insulin-dependent glucose disposal in the body; however, the effects of oxidative stress on insulin signaling and glucose transport activity in mammalian skeletal muscle are not well understood. We therefore studied the effects of a low-level in vitro oxidant stress (30–40μM H₂O₂) on basal and insulin-stimulated (5mU/ml) glucose transport activity and insulin signaling at 2, 4, and 6h in isolated rat soleus muscle. H₂O₂ increased basal glucose transport activity at 2 and 4h, but not at 6h. This low-level oxidant stress significantly impaired insulin-stimulated glucose transport activity at all time points, and was associated with inhibition of insulin-stimulated phosphorylation of Akt Ser⁴⁷³ and GSK-3β Ser⁹. In the presence of insulin, H₂O₂ decreased total protein expression of IRS-1 at 6h and IRS-2 at 4 and 6h. Phosphorylation of p38 MAPK Thr¹⁸⁰/Tyr¹⁸² was transiently increased by H₂O₂ in the presence and absence of insulin at 2 and 4h, but not at 6h. Selective inhibition of p38 MAPK with A304000 partially rescued the H₂O₂-induced reduction in insulin-stimulated glucose transport activity. These results indicate that direct in vitro exposure of isolated mammalian skeletal muscle to a low-level oxidant stress impairs distal insulin signaling and insulin-stimulated glucose transport activity, at least in part, due to a p38 MAPK-dependent mechanism.