Aim: We investigated the effect of elevated plasma free fatty acid and insulin concentrations on PDK4 mRNA transcript and protein content and long-chain acyl-CoAs (LCACs) accumulation in human skeletal muscle. Methods: On 2 occasions, 10 healthy men underwent hyperinsulinemic-euglycemic clamps for 6 h with (LIPID) and without (CON) intravenous Intralipid (20% at 90 ml/h) plus heparin (200 U prime + 600 U/h) infusion. Results: Glucose disposal was 50% lower at the end of the clamp in the LIPID compared to CON trial (37.8 ± 4.4 and 79.6 ± 4.0 µmol/kg lean mass. min, respectively, P < 0.01). In the LIPID trial, muscle LCACs concentration increased after 6 h, but not 3 h of lipid infusion (P < 0.01). Muscle PDK4 mRNA, but not protein, was downregulated by 2-fold within 3 h in both clamps, and decreased further (6-fold, P < 0.01) at 6 h in the CON, but not the LIPID clamp. The lipid-induced attenuation in the suppression of PDK4 gene expression was not dependent on the activation of the Akt/FOXO3 pathway. Conclusion: Accumulation of intramuscular lipids plays a more important role than impaired activation of Akt-mediated pathways in the regulation of muscle PDK4 gene expression in lipid-induced acute insulin resistant states.