Skeletal muscle insulin resistance is a major defect in Type 2 diabetes and the Metabolic Syndrome. We have evidence demonstrating that skeletal muscle becomes insulin resistant after 3wk of high-fat feeding (HFF; 42% energy from fat) in male C57BL/6J mice, as evidenced by impaired peripheral insulin sensitivity (~40% decrease vs. Chow) and reduced skeletal muscle glucose uptake during a hyperinsulinaemic-euglycaemic clamp in vivo. The factor(s) responsible for this skeletal muscle defect remain unclear, but an altered cellular redox state has been proposed to play a role. In line with this, skeletal muscle NADP⁺ levels were elevated (551±14 vs. 470±17pmol/mg for Chow, p<0.01) while NADPH:NADP⁺ was reduced (0.61±0.05 vs. 0.93±0.09, p<0.01) at 3wk of HFF (i.e. when muscle became insulin resistant). G6PD, which catalyses glucose-6-phosphate (G-6-P) for further metabolism and in doing so converts NADP⁺ to NAPDH, was reduced (11.7±0.3 vs. 14.3±0.8µmol/min/mg for Chow, p<0.05). Impaired G6PD has been linked to oxidative stress in non-muscle tissues, yet skeletal muscle GSH:GSSG levels, glutathione peroxidase activity, and NADPH oxidase gene expression were unaltered after 3wk of HFF. Impaired G6PD also leads to elevated G-6-P, a potent inhibitor of hexokinase (HK)II. Interestingly, whereas insulin increased HKII activity in Chow mice by 13±4%, this increase was ablated in HFF mice (-5±6%, p<0.05 vs. Chow). A regulatory role for G6PD in insulin-stimulated glucose uptake was confirmed in L6 muscle cells; incubation with the G6PD inhibitor 6-aminonicotinamide reduced G6PD activity 19±5% versus Control (p<0.02) and reduced insulin-stimulated glucose uptake 41±14% (p<0.05). Thus, G6PD regulates insulin-stimulated glucose uptake in skeletal muscle. This is not associated with enhanced oxidative stress, but rather impaired HKII activation. Our finding that impaired G6PD parallels the induction of insulin resistance in skeletal muscle indicates a potential role for G6PD in this metabolic defect.