OBJECTIVE-Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; encoded by Pck1) catalyzes the first committed step in gluconeogenesis. Extensive evidence demonstrates a direct correlation between PEPCK-C activity and glycemia control. Therefore, we aimed to evaluate the metabolic impact and their underlying mechanisms of knocking down hepatic PEPCK-C in a type 2 diabetic model. RESEARCH DESIGN AND METHODS-PEPCK-C gene targeting was achieved using adenovirus-transduced RNAi. The study assessed several clinical symptoms of diabetes and insulin signaling in peripheral tissues, in addition to changes in gene expression, protein, and metabolites in the liver. Liver bioener-getics was also evaluated. RESULTS-Treatment resulted in reduced PEPCK-C mRNA and protein. After treatment, improved glycemia and insulinemia, lower triglyceride, and higher total and HDL cholesterol were measured. Unsterified fatty acid accumulation was observed in the liver, in the absence of de novo lipogenesis. Despite hepatic lipidosis, treatment resulted in improved insulin signaling in the liver, muscle, and adipose tissue. O2 consumption measurements in isolated hepatocytes demonstrated unaltered mitochondrial function and a consequent increased cellular energy charge. Key regulatory factors (FOXO1, hepatocyte nuclear factor-4α, and peroxisome proliferator-activated receptor-γ coactivator [PGC]-1α) and enzymes (G6Pase) implicated in gluconeogenesis were downregulated after treatment. Finally, the levels of Sirt1, a redox-state sensor that modulates gluconeogenesis through PGC-1α, were diminished. CONCLUSIONS-Our observations indicate that silencing PEPCK-C has direct impact on glycemia control and energy metabolism and provides new insights into the potential significance of the enzyme as a therapeutic target for the treatment of diabetes. © 2008 by the American Diabetes Association.