Increasing evidence has suggested that dysregulation of microRNAs (miRNAs) could contribute to tumor progression. Previous miRNA microarray analysis illustrated that miR-335 is downregulated in various cancers; however, the role of miR-335 on hepatocellular carcinoma (HCC) has not been well elucidated. In this study, we investigated the biological functions and molecular mechanisms of miR-335 in human HCC in vitro, discussing whether it could be a therapeutic biomarker of HCC in the future. Four HCC cell lines and samples from 62 patients with HCC were analyzed for the expression of miR-335 by quantitative RT-PCR. Overexpression of miR-335 was established by transfecting mimics into HepG2 and HuH7 cells. Cell proliferation and cell migration were assessed by cell viability assay and transwell assay. Luciferase reporter assay and Western blot were to verify ROCK1 as a novel target gene of miR-335. We observed that miR-335 was downregulated in human HCC tissues and in all four HCC cell lines. The MTT assay revealed that overexpression of miR-335 subsequently inhibited cell growth. Furthermore, the transwell assay also showed significant cell migration inhibition in miR-335 transfectant. The expression of ROCK1 was decreased evidently after overexpression of miR-335, indicating that ROCK1 is a target gene for miR-335. Our data revealed that miR-335 could inhibit the proliferation and migration invasion of HCC cells via regulating ROCK1, suggesting that miR-335 could be a therapeutic biomarker of HCC in the future.