Objective: To investigate the role of Sonic Hedgehog (SHH) signaling pathway in proliferation of hepatocellular carcinoma (HCC) cells and the impacts of its inhibition on proliferation and chemosensitivity of HCC cells. Methods: Human HCC SMMC-7721 cells were respectively exposed to recombinant SHH N-terminus (rSHH-N), SHH neutralizing antibody (anti-SHH), and SHH pathway inhibitor cyclopamine for different time periods, and then the proliferation status of the cells was determined by MTT assay. The inhibitory effects on the proliferation of SMMC-7721 cells among 5-fluorouracil (5-FU), anti-SHH, cyclopamine, anti-SHH plus 5-FU and cyclopamine plus 5-FU were compared. Results: The proliferation of SMMC-7721 cells was significantly increased after rSHH-N treatment, while it was significantly decreased after anti-SHH or cyclopamine treatment, and all the effects presented a concentration- and time-dependent manner (all P<0.05). The inhibitory effect of anti-SHH plus 5-FU or cyclopamine plus 5-FU was significantly greater than any of the drugs used alone (all P<0.05). Conclusion: SHH signaling pathway plays an important role in growth of HCC cells, and blockage of SHH signaling pathway can inhibit proliferation of HCC cells and enhance their sensitivity to chemotherapeutic drugs.