Malonyl-coenzyme A (malonyl-CoA), generated by acetyl-CoA carboxylases ACC1 and ACC2, is a key metabolite in the regulation of energy homeostasis. Here, we show that Acc2-/- mutant mice have a normal life span, a higher fatty acid oxidation rate, and lower amounts of fat. In comparison to the wild type, Acc2-deficient mice had 10- and 30-fold lower levels of malonyl-CoA in heart and muscle, respectively. The fatty acid oxidation rate in the soleus muscle of the Acc2-/- mice was 30% higher than that of wild-type mice and was not affected by addition of insulin; however, addition of insulin to the wild-type muscle reduced fatty acid oxidation by 45%. The mutant mice accumulated 50% less fat in their adipose tissue than did wild-type mice. These results raise the possibility that pharmacological manipulation of ACC2 may lead to loss of body fat in the context of normal caloric intake.