In diabetic patients, α-lipoic acid (LA) improves skeletal muscle glucose transport, resulting in increased glucose disposal; however, the molecular mechanism of action of LA is presently unknown. We studied the effects of LA on basal and insulin-stimulated glucose transport in cultured rat L6 muscle cells that overexpress GLUT4. When 2-deoxy-D-glucose uptake was measured in these cells, they were more sensitive and responsive to insulin than wild-type L6 cells. LA, at concentrations ≤1 mmol/l, had only small effects on glucose transport in cells not exposed to oxidative stress. When cells were exposed to glucose oxidase and glucose to generate H₂O₂ and cause oxidative stress, there was a marked decrease in insulin-stimulated glucose transport. Pretreatment with LA over the concentration range of 10-1,000 μmol/l protected the insulin effect from inhibition by H₂O₂. Both the R and S isomers of LA were equally effective. In addition, oxidative stress caused a significant decrease (∼50%) in reduced glutathione concentration, along with the rapid activation of the stress-sensitive p38 mitogen-activated protein kinase. Pretreatment with LA prevented both of these events, coincident with protecting insulin action. These studies indicate that in muscle, the major site of insulin-stimulated glucose disposal, one important effect of LA on the insulin-signaling cascade is to protect cells from oxidative stress-induced insulin resistance.