Objective: To observe the inhibitory effect of RNA interference (RNAi) on the expression of nodal gene in human hepatocellular carcinoma (HCC) cells, and explore the feasibility of nodal gene targeting in HCC gene therapy. Methods: SMMC-7721 cells were transfected with the plasmid vector containing a short hairpin RNA (shRNA) sequence targeting the nodal gene. After assessing the transfection efficiency, the nodal gene and protein expression was detected by real-time quantitive PCR and Western blot,respectively. Furthermore, the effect of nodal gene interference on the proliferation of SMMC-7721 cells in vitro was studied. Results: The plasmid containing shRNA had excellent transfection efficiency in SMMC-7721 cells. After transfection, the expression of nodal mRNA and protein of SMMC-7721 cells was significantly inhibited, and their proliferation in vitro was also significantly suppressed. Conclusion: The nodal gene and protein expression as well as cell proliferation of HCC cells can be effectively inhibited by RNAi technique, so nodal gene may be a potential therapeutic target for HCC.