Under various conditions, mammals have the ability to maintain serum glucose concentration within a narrow range. SIRT1 plays an important role in regulating gluconeogenesis and fat metabolism; however, the underlying mechanisms remain elusive. Here, we show that SIRT1 forms a complex with FOXO3a and NRF1 on the SIRT6 promoter and positively regulates expression of SIRT6, which, in turn, negatively regulates glycolysis, triglyceride synthesis, and fat metabolism by deacetylating histone H3 lysine 9 in the promoter of many genes involved in these processes. Liver-specific deletion of SIRT6 in mice causes profound alterations in gene expression, leading to increased glycolysis, triglyceride synthesis, reduced β oxidation, and fatty liver formation. Human fatty liver samples exhibited significantly lower levels of SIRT6 than did normal controls. Thus, SIRT6 plays a critical role in fat metabolism and may serve as a therapeutic target for treating fatty liver disease, the most common cause of liver dysfunction in humans. © 2010 Elsevier Inc.