Hepatocellular carcinoma is the primary malignancy of the liver and is the third leading cause of cancer-related death worldwide. Despite its severity, the treatment methods for hepatocellular carcinoma are limited due to poor prognosis. This study identified the short hairpin RNA-mediated silencing of myosin VI as a potential approach for the treatment of hepatocellular carcinoma. Firstly, the expression of myosin VI was analyzed in 57 hepatocellular carcinoma samples and 10 non-neoplastic samples by immunohistochemistry. The results demonstrated that myosin VI expression was much stronger in hepatocellular carcinoma tissues than in adjacent normal tissues. Myosin VI short hairpin RNA was then transduced into HepG2 and SMMC-7721 hepatocellular carcinoma cell lines, respectively, using a lentivirus delivery system. Knockdown of myosin VI led to a significant reduction in cell proliferation and colony-forming capacity, as well as a blockade of cell cycle progression. Moreover, an obvious decrease in PRAS40 phosphorylation and a concomitant increase in p38 phosphorylation were observed in myosin VI knockdown cells, which suggest that myosin VI silencing inhibits hepatocellular carcinoma cell growth invitro probably via inactivation of PRAS40 and activation of p38 mitogen-activated protein kinase-dependent signaling pathway. This study highlights the potential of myosin VI to be developed as a target for hepatocellular carcinoma therapy.