Insulin signaling in the liver blunts glucose production and stimulates triglyceride biosynthesis. FoxO1 is required for cAMP induction of hepatic glucose production and is permissive for the effect of insulin to suppress this process. Moreover, FoxO1 ablation increases lipogenesis. In this study, we investigated the pleiotropic actions of FoxO1 on glucose and lipid metabolism. To this end, we reconstituted FoxO1 function in mice with a liver-specific deletion of Foxo1 using targeted knock-in of an allele encoding a DNA binding–deficient FoxO1 mutant (L-DBD). Chow-reared L-DBD mice showed defects in hepatic glucose production but normal liver triglyceride content despite increased rates of de novo lipogenesis and impaired fatty acid oxidation in isolated hepatocytes. Gene expression studies indicated that FoxO1 regulates the expression of glucokinase via a cell-nonautonomous coregulatory mechanism, while its regulation of glucose-6-phosphatase proceeds via a cell-autonomous action as a direct transcriptional activator. These conclusions support a differential regulation of hepatic glucose and lipid metabolism by FoxO1 based on the mechanism by which it alters the expression of key target genes involved in each process.