The sekelsky mothers against dpp3 (Smad3) functions as a transcriptional modulator activated by transforming growth factor-β (TGF-β). Accumulated evidences indicated that Smad3 played the important roles in carcinogenesis and progression of hepatocellular carcinoma (HCC). Up to now, the regulatory mechanism of Smad3 in HCC still remains unclear. It has been known that some particular microRNAs (miRNAs) involve in carcinogenesis through the regulation of gene expressions with targeting mRNAs. In our study, the unknown candidates of miRNAs that target Smad3 mRNA were searched by using a newly established in vivo approach, the miRNA in vivo precipitation (miRIP). Using a loss-of-function assay, we demonstrated that miR-17 directly targeted Smad3 in HCC cells and inhibition on miR-17 increased Smad3 expression. Furthermore, we found that downregulation on Smad3 expression was consistent with high level of miR-17 in HCC tissues of patients when compared with around normal liver tissues. The manipulated miR-17 silence in HCC cells suppressed their growth of both in vitro and in vivo. Such suppression on cell growth could be recovered through downregulating Smad3. In addition, miR-17 affected cell proliferation through arresting cell cycle in G1 phase. The negative correlation between levels of miR-17 and protein levels of Smad3 was supported by the results of analysis with HCC tissue chip. In summary, for the first time, we confirmed that miR-17 directly targeted Smad3 mRNA and downregulated Smad3 protein expression in HCC. Our results indicated that the increased expression of miR-17 promoted carcinogenesis of HCC through down-regulations of Smad3, suggesting miR-17 might serve as the potential diagnostic and therapeutic targets for clinical HCC.