Earlier reports suggest a link between mitochondrial dysfunction and development of hepatic insulin resistance. Here we used a murine model heterozygous (HET) for a mitochondrial trifunctional protein (MTP) gene defect to determine if a primary defect in mitochondrial long-chain fatty acid oxidation disrupts hepatic insulin action. Hyperinsulinemic-euglycemic clamps and signaling studies were performed for assessment of whole-body and hepatic insulin resistance/signaling. In addition, hepatic fatty acid oxidation and hepatic insulin action were assessed in vitro using primary hepatocytes isolated from HET and wildtype (WT) mice. In both hepatic mitochondria and isolated primary hepatocytes, heterozygosity of MTP caused an ∼50% reduction in mitochondrial fatty acid oxidation, a significantly impaired glucose disposal during the insulin clamp, and a markedly lower insulin-stimulated suppression of hepatic glucose production. HET mice also exhibited impaired insulin signaling, with increased hepatic phosphorylation of IRS2 (ser731) and reduced Akt phosphorylation (ser473) in both hepatic tissue and isolated primary hepatocytes. Assessment of insulin-stimulated FOXO1/phospho-FOXO1 protein content and PEPCK/G6Pase messenger RNA (mRNA) expression did not reveal differences between HET and WT mice. However, insulin-induced phosphorylation of GSK3β was significantly blunted in HET mice. Hepatic insulin resistance was associated with an increased methylation status of the catalytic subunit of protein phosphatase 2A (PP2A-C), but was not associated with differences in hepatic diacylglycerol content, activated protein kinase C-ϵ (PKC-ϵ), inhibitor κB kinase β (IKK-β), c-Jun N-terminal kinase (JNK), or phospho-JNK protein contents. Surprisingly, hepatic ceramides were significantly lower in the HET mice compared with WT. Conclusion: A primary defect in mitochondrial fatty acid β-oxidation causes hepatic insulin resistance selective to hepatic glycogen metabolism that is associated with elevated methylated PP2A-C, but independent of other mechanisms commonly considered responsible for insulin resistance. (HEPATOLOGY 2013;)