Micro-RNAs (miRNAs) have been suggested to play pivotal roles in multifarious diseases associated with the posttranscriptional regulation of protein-coding genes. In this study, we aimed to investigate the function of miRNAs in type 2 diabetes mellitus. The miRNAs expression profiles were examined by miRNA microarray analysis of skeletal muscles from healthy and Goto-Kakizaki rats. We identified four up-regulated miRNAs, and 11 miRNAs that were down-regulated relative to normal individuals. Among induced miRNAs were three paralogs of miR-29, miR-29a, miR-29b, and miR-29c. Northern blotting further confirmed their elevated expression in three important target tissues of insulin action: muscle, fat, and liver of diabetic rats. Adenovirus-mediated overexpression of miR-29a/b/c in 3T3-L1 adipocytes could largely repress insulin-stimulated glucose uptake, presumably through inhibiting Akt activation. The increase in miR-29 level caused insulin resistance, similar to that of incubation with high glucose and insulin in combination, which, in turn, induced miR-29a and miR-29b expression. In this paper, we demonstrate that Akt is not the direct target gene of miR-29 and that the negative effects of miR-29 on insulin signaling might be mediated by other unknown intermediates. Taken together, these data reveal the crucial role of miR-29 in type 2 diabetes.