Obesity is risk factor for the development of serious diseases including non-alcoholic fatty liver disease and type 2 diabetes, and dysregulated lipid metabolism is a common feature of these disorders. The perilipin (Plin) family of proteins are highly expressed proteins that associate with intracellular lipid droplets and regulate lipid metabolism, particularly in oxidative tissues where it is highly expressed. The aim of this study was to investigate the role of Plin5 in regulating liver lipid metabolism. PLIN5 liver-specific knockout mice (PLIN5-LKO) were generated by crossing PLIN5 flox/flox mice with albumin-Cre mice. Primary hepatocytes were isolated by collagenase digestion and were cultured and lipid metabolism was assessed by radiometric methods. Plin5 ablation decreased free fatty acid oxidation by 35% and intrahepatic triglyceride-derived fatty acid oxidation by 69%. Fatty acid uptake and storage rates into triglyceride were decreased by 40% in PLIN5-LKO hepatocytes compared with wild-type controls and de novo lipogenesis was increased by 76%. Notably, triglyceride secretion from hepatocytes was increased by 83%. Hence, PLIN5 results in marked remodeling of lipid metabolism in hepatocytes. In vivo studies show that PLIN5-LKO mice have normal body mass, body composition, whole-body substrate oxidation rates, food intake and blood lipid profiles. Despite having lower hepatic triglyceride levels, PLIN5-LKO mice are glucose intolerant. On-going hyperinsulinemic-euglycemic clamp studies are determining whether this is due to reduced insulin action. In conclusion PLIN5 is essential for maintaining normal lipid homeostasis in the liver and its deletion leads to marked glucose intolerance in mice.