Triglyceride synthesis in most mammalian tissues involves the sequential addition of fatty acids to a glycerol backbone, with unique enzymes required to catalyze each acylation step. Acylation at the sn-2 position requires 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) activity. To date, seven Agpat genes have been identified based on activity and/or sequence similarity, but their physiological functions have not been well established. We have generated a mouse model deficient in AGPAT6, which is normally expressed at high levels in brown adipose tissue (BAT), white adipose tissue (WAT), and liver. Agpat6-deficient mice exhibit a 25% reduction in body weight and resistance to both diet-induced and genetically induced obesity. The reduced body weight is associated with increased energy expenditure, reduced triglyceride accumulation in BAT and WAT, reduced white adipocyte size, and lack of adipose tissue in the subdermal region. In addition, the fatty acid composition of triacylglycerol, diacylglycerol, and phospholipid is altered, with proportionally greater polyunsaturated fatty acids at the expense of monounsaturated fatty acids. Thus, Agpat6 plays a unique role in determining triglyceride content and composition in adipose tissue and liver that cannot be compensated by other members of the Agpat family.