Growing evidence indicates that deregulation of microRNAs (miRNAs) contributes to tumorigenesis. Down‐regulation of miR‐195 has been observed in various types of cancers. However, the biological function of miR‐195 is still largely unknown. In this study we aimed to elucidate the pathophysiologic role of miR‐195. Our results showed that miR‐195 expression was significantly reduced in as high as 85.7% of hepatocellular carcinoma (HCC) tissues and in all of the five HCC cell lines examined. Moreover, introduction of miR‐195 dramatically suppressed the ability of HCC and colorectal carcinoma cells to form colonies in vitro and to develop tumors in nude mice. Furthermore, ectopic expression of miR‐195 blocked G₁/S transition, whereas inhibition of miR‐195 promoted cell cycle progression. Subsequent investigation characterized multiple G₁/S transition‐related molecules, including cyclin D1, CDK6, and E2F3, as direct targets of miR‐195. Silencing of cyclin D1, CDK6, or E2F3 phenocopied the effect of miR‐195, whereas overexpression of these proteins attenuated miR‐195‐induced G₁ arrest. In addition, miR‐195 significantly repressed the phosphorylation of Rb as well as the transactivation of downstream target genes of E2F. These results imply that miR‐195 may block the G₁/S transition by repressing Rb‐E2F signaling through targeting multiple molecules, including cyclin D1, CDK6, and E2F3. Conclusion: Our data highlight an important role of miR‐195 in cell cycle control and in the molecular etiology of HCC, and implicate the potential application of miR‐195 in cancer therapy. (HEPATOLOGY 2009.)