Glycerol-3-phosphate acyltransferase (GPAT) controls the first step of triglyceride (TAG) synthesis. Three distinct GPAT activities have been identified, two localized in mitochondria and one in microsomes. Mitochondrial GPAT1 (mtGPAT1) is abundantly expressed in the liver and constitutes approximately 50% of total GPAT activities in this organ. Hepatic mtGPAT1 activity is elevated in obese rodents. Mice deficient in mtGPAT1 have an improved lipid profile. To investigate if beneficial effects can result from reduced hepatic expression of mtGPAT1 in adult obese mice, adenoviral vector-based short hairpin RNA interference (shRNA) technology was used to knockdown mtGPAT1 expression in livers of ob/ob mice. Reduced expression of mtGPAT1 mRNA in liver of ob/ob mice resulted in dramatic and dose dependent reduction in mtGPAT1 activity. Reduced hepatic TAG, diacylglycerol, and free fatty acid, as well as reduced plasma cholesterol and glucose, were also observed. Fatty acid composition analysis revealed decrease of C16:0 in major lipid species. Our results demonstrate that acute reduction of mtGPAT1 in liver of ob/ob mice reduces TAG synthesis, which points to a role for mtGPAT1 in the correction of obesity and related disorders.