Glucose-6-phosphatase (G6Pase) is a key enzyme that is responsible for the production of glucose in the liver during fasting or in type 2 diabetes mellitus (T2DM). During fasting or in T2DM, peroxisome proliferator-activated receptor α (PPARα) is activated, which may contribute to increased hepatic glucose output. However, the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in these states is not well understood. We evaluated the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in fasting and T2DM states. In PPARα-null mice, both hepatic G6Pase and phosphoenolpyruvate carboxykinase levels were not increased in the fasting state. Moreover, treatment of primary cultured hepatocytes with Wy14,643 or fenofibrate increased the G6Pase mRNA level. In addition, we have localized and characterized a PPAR-responsive element in the promoter region of the G6Pase gene. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα binding to the putative PPAR-responsive element of the G6Pase promoter was increased in fasted wild-type mice and db/db mice. These results indicate that PPARα is responsible for glucose production through the up-regulation of hepatic G6Pase gene expression during fasting or T2DM animal models.