CCAAT/enhancer-binding protein-α (C/EBPα) is a member of the basic leucine zipper transcription factor family and regulates expression of several enzymes in the liver that control glucose and lipid metabolism. Using adenovirus-transduced silent interfering (si)RNA against C/EBPα, endogenous liver C/EBPα protein was knocked down by 70–80% in 8-wk-old wild-type (WT) and db/db mice. In WT mice, fasting blood glucose concentrations were reduced approximately 24% without changes in plasma free fatty acid and triglycerides, when compared with LacZ adenovirus-treated control mice. Ad-C/EBPα siRNA treatment nearly normalized fasting glucose and significantly reduced plasma insulin and free fatty acid content, even though there was no elevation of C/EBPα protein in the livers of db/db mice. In parallel with the changes in glucose levels, hepatic glucose production was significantly reduced in C/EBPα siRNA-treated WT and db/db mice. mRNA levels of phyosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and liver glycogen synthase were decreased in the C/EBPα siRNA-treated WT and db/db mice. Interestingly, the magnitude of reduction in these enzymes was more profound in db/db mice. C/EBPα siRNA also decreased mRNA levels of proliferator activator protein-γ coactivator-1α in both the WT and db/db mice but reduced cAMP response element-binding protein only in WT and did not alter hepatic nuclear factor-4α and CBP/p300 expression. Expression of genes involved in lipogenesis, such as fatty acid synthase, acetyl-CoA carboxylase, and sterol regulatory element-binding protein-1c was robustly suppressed in the C/EBPα siRNA-treated db/db mice. Taken together, these results indicate that C/EBPα plays an important role in maintaining glucose and lipid homeostasis in the liver.