Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first step in the synthesis of glycerolipids and glycerophospholipids. Microsomal GPAT, the major GPAT activity, is encoded by at least two closely related genes, GPAT3 and GPAT4. To investigate the in vivo functions of GPAT3, we generated Gpat3-deficient mice (Gpat3^-/-). Total GPAT activity in white adipose tissue of Gpat3^-/- mice was reduced by 80%, suggesting that GPAT3 is the predominant GPAT in this tissue. In liver, GPAT3-deletion had no impact on total GPAT activity, but resulted in a 30% reduction in N-ethylmaleimide-sensitive GPAT activity. The Gpat3^-/- mice were viable and fertile, and exhibited no obvious metabolic abnormalities on standard laboratory chow. However, when fed a high-fat diet, female Gpat3^-/- mice showed decreased body weight gain and adiposity and increased energy expenditure. Increased energy expenditure was also observed in male Gpat3^-/- mice, although it was not accompanied by a significant change in body weight. GPAT3-deficiency lowered fed, but not fasted, glucose levels, and tended to improve glucose tolerance in diet-induced obese male and female mice. On a high-fat diet, Gpat3^-/- mice had enlarged livers and displayed a dysregulation in cholesterol metabolism. These data establish GPAT3 as the primary GPAT in white adipose tissue and reveal an important role of the enzyme in regulating energy, glucose, and lipid homeostasis.