Glycerol, a product of adipose tissue lipolysis, is an important substrate for hepatic glucose synthesis. However, little is known about the regulation of hepatic glycerol metabolism. Here we show that several genes involved in the hepatic metabolism of glycerol, i.e., cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase (GPDH), glycerol kinase, and glycerol transporters aquaporin 3 and 9, are upregulated by fasting in wild-type mice but not in mice lacking PPARα. Furthermore, expression of these genes was induced by the PPARα agonist Wy14643 in wild-type but not PPARα-null mice. In adipocytes, which express high levels of PPARγ, expression of cytosolic GPDH was enhanced by PPARγ and β/δ agonists, while expression was decreased in PPARγ+/− and PPARβ/δ−/− mice. Transactivation, gel shift, and chromatin immunoprecipitation experiments demonstrated that cytosolic GPDH is a direct PPAR target gene. In line with a stimulating role of PPARα in hepatic glycerol utilization, administration of synthetic PPARα agonists in mice and humans decreased plasma glycerol. Finally, hepatic glucose production was decreased in PPARα-null mice simultaneously fasted and exposed to Wy14643, suggesting that the stimulatory effect of PPARα on gluconeogenic gene expression was translated at the functional level. Overall, these data indicate that PPARα directly governs glycerol metabolism in liver, whereas PPARγ regulates glycerol metabolism in adipose tissue.