It has been reported that Stat5 is overexpressed in a variety of human cancer cell lines and primary tumors. Inhibition of Stat5 in tumor cell lines has been associated with growth suppression and induction of apoptosis. However, no one of published studies have investigated the expression and role of Stat5 in hepatocellular carcinoma. In this study, we used human hepatocellular carcinoma cell line SMMC7721 as a model to demonstrate that Stat5 was highly expressed in these cells. Next we showed that RNAi mediated Stat5 knockdown could inhibit the proliferation and induce the apoptosis of SMMC7721 cells in vitro. Furthermore, we demonstrated that Stat5 knockdown inhibited the growth and induced the apoptosis of SMMC7721 cells in xenografts in nude mice. Taken together, our in vitro and in vivo data suggest that Stat5 plays an important role in human hepatocellular carcinoma. Inhibition of Stat5 by RNAi holds promise to be a novel gene therapy vector for hepatocellular carcinoma.