Accumulation of triglycerides (TG) in the liver is generally associated with hepatic insulin resistance. We questioned whether acute hepatic steatosis induced by pharmacological blockade of -oxidation affects hepatic insulin sensitivity, i.e., insulin-mediated suppression of very low density lipoprotein (VLDL) production and insulin-induced activation of PI3- kinase and protein kinase B (PKB). Tetradecylglycidic acid (TDGA), an inhibitor of carnitine palmitoyl transferase-1 (CPT1), was used for this purpose. Male C57BL/6J mice received 30 mg/kg TDGA or its solvent intraperitoneally and were subsequently fasted for 12 hours. CPT1-inhibition resulted in severe microvesicular hepatic steatosis (19.9 ± 8.3 vs. 112.4 ± 25.2 nmol TG/mg liver, control vs. treated, P<0.05) with elevated plasma non-esterified fatty acid (0.68 ± 0.25 vs. 1.21 ± 0.41 mM, P<0.05) and plasma TG (0.39 ± 0.16 vs. 0.60 ± 010 mM, p<0.05) concentrations. VLDL-TG production rate was not affected upon CPT1- inhibition (74.9 ± 15.2 vs. 79.1 ± 12.8 µmol TG/kg/min, control vs. treated), although treated mice secreted larger VLDL particles (59.3 ± 3.6 vs. 66.6 ± 4.5 nm diameter, p<0.05). Infusion of insulin under euglycemic conditions suppressed VLDL production rate in control and treated mice by 43 and 54%, respectively, with formation of smaller VLDL particles (51.2 ± 2.5 and 53.2 ± 2.8 nm diameter). Insulin-induced IRS1- and IRS2- associated PI3K activity and PKB-phosphorylation were not affected upon TDGA treatment. In conclusion, acute hepatic steatosis caused by pharmacological inhibition of -oxidation is not associated with reduced hepatic insulin sensitivity, indicating that hepatocellular fat content per se is not causally related to insulin resistance.