MicroRNAs (miRNAs) are approximately 22‐nucleotide noncoding RNAs that constitute silencers of target gene expression. Aberrant expression of miRNA has been linked to a variety of cancers, including hepatocellular carcinoma (HCC). Hepatitis C virus (HCV) infection is considered a major cause of chronic liver disease and HCC, although the mechanism of virus infection–associated hepatocarcinogenesis remains unclear. We report a direct role of miRNAs induced in HCV‐infected primary human hepatocytes that target the tumor suppressor gene DLC‐1 (a Rho GTPase‐activating protein), which is frequently deleted in HCC, and other solid human tumors. MicroRNA miR‐141 that targets DLC‐1 was accentuated in cells infected with HCV genotypes 1a, 1b, and 2a. We present several lines of evidence that efficient HCV replication requires miR‐141–mediated suppression of DLC‐1. An increase in miR‐141 correlated with the inhibition of DLC‐1 protein in HCV‐infected cells. Depletion of miR‐141 with oligonucleotides complementary to the miRNAs inhibited virus replication, whereas artificially increased levels of intracellular miR‐141 enhanced HCV replication. HCV‐infected hepatocytes showed enhanced cell proliferation that can be countered by overexpression of DLC‐1. Conclusion: The collective results of this study suggest a novel mechanism of HCV infection–associated miRNA‐mediated regulation of a tumor suppressor protein that has the ability to influence cell proliferation and HCV infection–mediated liver cancer. (HEPATOLOGY 2011)