14-kDa Phosphohistidine phosphatase (PHP14), the first histidine phosphatase protein identified in vertebrates, was recently revealed to play an essential role in lung cancer. The function of this gene in other tumors is unclear; however, in this study, we demonstrate that PHP14 is highly expressed in hepatocellular carcinoma (HCC) tissues and cell lines compared with adjacent non-cancerous human liver tissues and cells (P<0.05). We used lentivirus-mediated delivery of small interfering RNA (siRNA) to knockdown the expression of PHP14 in an HCC cell line and investigate the effects of PHP14 on cell growth in vitro. Cell proliferation was inhibited and cell apoptosis was significantly increased. PHP14-siRNA affected the cell cycle and promoted G1→S phase transition in HCC cells. These results demonstrate that the knockdown of PHP14 expression by lentivirus-delivered siRNA may be a useful therapeutic approach for the treatment of HCC.