Background Oxidative stress-induced reactive oxygen species are associated with the clinical manifestation of insulin resistance. Evidence suggests that antioxidant treatment may reduce this incidence. Aim of the study This study determined whether glucose oxidase (GO)-induced insulin resistance in cultured skeletal muscle cells could be ameliorated by pre-treatment with gamma-tocopherol (GT). Methods Insulin sensitivity in L6 myotubes was assessed by 2-deoxy-d-[3H]-glucose uptake. The phosphorylation of distal insulin signaling proteins Akt and the Akt substrate AS160 were determined by western blot. Results One hour treatment with 100 mU/ml GO decreased insulin-stimulated glucose uptake (P < 0.001). Pre-treatment with GT either partially (100 卩 or completely (200 卩 restored insulin-stimulated glucose uptake in cells after GO-induced insulin resistance. GO-induced oxidative stress did not impair insulin stimulated phosphorylation of Akt or AS160, but 200 占GT increased insulin-stimulated phosphorylation of these key signaling proteins (P < 0.05). Conclusions High-dose (200 卩 GT treatment ameliorated oxidative stress-induced insulin resistance in cultured rat L6 skeletal muscle cells.