To understand the mechanisms in lipid-induced insulin resistance, a more physiologic approach is to enhance FA availability through the diet. Nine healthy men ingested two hypercaloric diets (+75 E%) for three days, enriched in unsaturated FA (78 E% fat; UNSAT) or carbohydrates (80 E% carbohydrate; CHO) as well as a eucaloric control diet (CON). Compared to CON, UNSAT reduced whole body and leg glucose disposal during a hyperinsulinemic-euglycemic clamp, while decreasing hepatic glucose production. In muscle, DAG and IMTG were increased. The accumulated DAG was sn-1,3 DAG, which is known not to activate PKC, and insulin signaling was intact. UNSAT decreased PDH-E1α protein content, and increased inhibitory PDH-E1α Ser(300) phosphorylation and FA oxidation. CHO increased whole body and leg insulin sensitivity, while increasing hepatic glucose production. After CHO, muscle PDH-E1α Ser(300) phosphorylation was decreased, and glucose oxidation increased. After UNSAT, but not CHO, muscle G6P content was 103 % higher compared to CON during the clamp. Thus, PDH-E1α expression and covalent regulation, and hence the TCA influx of pyruvate-derived acetyl-CoA relative to beta-oxidation-derived acetyl-CoA, are suggested to impact on insulin-stimulated glucose uptake. Taken together, the oxidative metabolic fluxes of glucose and FA are powerful and opposite regulators of insulin action in muscle.