Background & Aims: The expression levels of microRNA-29 (miR-29) family members (miR-29a, miR-29b, miR-29c, here denoted collectively as miR-29a-c) are increased in livers of Goto-Kakizaki diabetic rats and db/db diabetic mice. However, the functional consequences of miR-29a-c upregulation in diabetic livers are not explored. The objective of this study was to evaluate the roles of miR-29a-c in the regulation of hepatic glucose production and blood glucose levels using different mouse models. Methods: db/m, db/db diabetic and diet-induced obese (DIO) mice were injected with adenovirus expressing miR-29a-c through the tail vein. Blood glucose levels were measured and glucose-tolerance tests and pyruvate-tolerance tests were performed. To explore the molecular mechanism by which miR-29a-c regulate hepatic glucose metabolism, gain or loss of miR-29a-c function studies were performed in primary mouse hepatocytes and the direct effectors of miR-29-mediated effects on glucose metabolism were identified. Results: Adenovirus-mediated overexpression of miR-29a-c in the livers of db/m, db/db, and DIO mice decreased fasting blood glucose levels and improved glucose tolerance. Overexpression of miR-29a-c in primary hepatocytes and mouse livers decreased the protein levels of PGC-1 alpha and G6Pase, the direct targets of miR-29a-c, thereby reducing cellular, and hepatic glucose production. In contrast, loss of miR-29a-c function in primary hepatocytes increased the protein levels of PGC-1 alpha and G6Pase and increased cellular glucose production. Finally, enforced expression of PGC-1 alpha increased miR-29a-c expression levels in primary hepatocytes, thus forming a negative feedback regulation loop. Conclusions: miR-29a-c can regulate hepatic glucose production and glucose tolerance in mice. (C) 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.