Nonalcoholic fatty liver disease (NAFLD) and insulin resistance have recently been found to be associated with increased plasma concentrations of apolipoprotein CIII (APOC3) in humans carrying single nucleotide polymorphisms within the insulin response element of the APOC3 gene. To examine whether increased expression of APOC3 would predispose mice to NAFLD and hepatic insulin resistance, human APOC3 overexpressing (ApoC3Tg) mice were metabolically phenotyped following either a regular chow or high-fat diet (HFD). After HFD feeding, ApoC3Tg mice had increased hepatic triglyceride accumulation, which was associated with cellular ballooning and inflammatory changes. ApoC3Tg mice also manifested severe hepatic insulin resistance assessed by a hyperinsulinemic-euglycemic clamp, which could mostly be attributed to increased hepatic diacylglycerol content, protein kinase C-Ξ΅ activation, and decreased insulin-stimulated Akt2 activity. Increased hepatic triglyceride content in the HFD-fed ApoC3Tg mice could be attributed to a β70% increase in hepatic triglyceride uptake and β50% reduction hepatic triglyceride secretion. Conclusion: These data demonstrate that increase plasma APOC3 concentrations predispose mice to diet-induced NAFLD and hepatic insulin resistance. (Hepatology 2011;54:1650-1660)