Glucolipotoxicity plays an important role in the development and progression of type 2 diabetes. Impaired insulin secretion in vivo coincides with major alterations in carbohydrate and lipid metabolism in β-cells. Previously our group reported overexpression of PGC-1α by glucolipotoxicy especially in liver and pancreas might play a major role of glucolipotoxicity-induced beta-cell dysfunction and death. We searched for new miRNAs that could control the overexpression of PGC-1α in both β -cells and hepatocytes with the hypothesis that one miRNA could control the same target in multiple tissues. To search for miRNAs , we analyzed miRNA expression patterns in primary rat islets and hepatocytes and finally found a miRNA-374. Delivery of miR-374 significantly suppressed PGC-1α overexpression and improved glucotoxicity-induced β-cell dysfunction and hepatic glucose overproduction. Specific binding of miR-374 on the 3'-untranslated region (3'-UTR) of PGC-1α was confirmed by luciferase activity assay combined with mutational analysis. Injection of Ad-miR-374 in seven-week-old db/db mice remarkably improved glucose-stimulated insulin secretion and suppressed hepatic glucose overproduction by normalizing the gene expression in islets and hepatocytes altered by glucotoxicity. Here, we demonstrated that miR-374 could be a novel treatment target because it could simultaneously correct the glucotoxicityinduced dysfunction in both β-cells and hepatocytes in an animal model of T2DM.