De novo lipogenesis is an energy-expensive process whose role in adult mammals is poorly understood. We generated mice with liver-specific inactivation of fatty-acid synthase (FAS), a key lipogenic enzyme. On a zero-fat diet, FASKOL (FAS knockout in liver) mice developed hypoglycemia and fatty liver, which were reversed with dietary fat. These phenotypes were also observed after prolonged fasting, similarly to fasted PPARα-deficiency mice. Hypoglycemia, fatty liver, and defects in expression of PPARα target genes in FASKOL mice were corrected with a PPARα agonist. On either zero-fat or chow diet, FASKOL mice had low serum and hepatic cholesterol levels with elevated SREBP-2, decreased HMG-CoA reductase expression, and decreased cholesterol biosynthesis; these were also corrected with a PPARα agonist. These results suggest that products of the FAS reaction regulate glucose, lipid, and cholesterol metabolism by serving as endogenous activators of distinct physiological pools of PPARα in adult liver.