Triglycerides (or triacylglycerols) represent the major form of stored energy in eukaryotes. Triglyceride synthesis has been assumed to occur primarily through acyl CoA: diacylglycerol transferase (Dgat), a microsomal enzyme that catalyses the final and only committed step in the glycerol phosphate pathway 1, 2, 3. Therefore, Dgat has been considered necessary for adipose tissue formation and essential for survival. Here we show that Dgat-deficient (Dgat−/−) mice are viable and can still synthesize triglycerides. Moreover, these mice are lean and resistant to diet-induced obesity. The obesity resistance involves increased energy expenditure and increased activity. Dgat deficiency also alters triglyceride metabolism in other tissues, including the mammary gland, where lactation is defective in Dgat−/− females. Our findings indicate that multiple mechanisms exist for triglyceride synthesis and suggest that the selective inhibition of Dgat-mediated triglyceride synthesis may be useful for treating obesity.