MicroRNA (miRNA) is a class of endogenous small noncoding RNA that negatively regulates gene expression at the post-transcriptional level and plays an important role in the pathogenesis of various diseases. However, the identity and role of miRNAs involved in the development of insulin resistance resulting from mitochondrial dysfunction are largely unknown. In this study, mitochondrial dysfunction by genetic or metabolic inhibition induced an impairment of insulin signaling in SK-Hep1 cells via a reduction in the expression of IRS-1 protein. Significant up-regulation of miR-96, which was presumed to target IRS-1 3'UTR, was found in SK-Hep1 cells with mitochondrial dysfunction. Using reporter gene assay we confirmed that miR-96 authentically targeted IRS-1 3'UTR. Furthermore, the ectopic expression of miR-96 caused a substantial decrease in IRS-1 protein expression, and a consequent impairment in insulin signaling. These findings suggest that the up-regulation of miR-96 by mitochondrial dysfunction contributes to the development of insulin resistance by targeting IRS-1 in SK-Hep1 cells. (C) 2013 Elsevier Inc. All rights reserved.