Objective To construct recombinant adenovirus harboring snake venom cystatin (Ad/sv-cystatin) and to investigate its effect on invasion and metastasis (in vivo and in vitro) of human hepatocellular carcinoma (HCC) MHCC97H cells. Methods The recombinant Ad/sv-cystatin harboring sv-cystatin was constructed to infect MHCC97H cells. Then the growth of MHCC97H cells was assessed by CCK-8. Transwell matrigel assay was used to assess MHCC97H cell migration and invasiveness in vitro. Spontaneous lung metastasis assays were used to examine the effects of Ad/sv-cystatin on the invasion and metastasis of MHCC97H cells in vivo. Results Recombinant Ad/sv-cystatin harboring sv-cystatin gene could infect MHCC97H cells. Ad/sv-cystatin significantly inhibited the growth, migration and invasion of MHCC97H cells in vitro compared with control and Ad/null groups. Intra-tumoral injection of Ad/sv-cystatin significantly inhibited the lung metastasis of MHCC97H cells in nude mice compared with control and Ad/null-treated mice. Conclusion It is indicated that recombinant Ad/sv-cystatin can suppress MHCC97H cell growth, invasion and metastasis in vitro and in vivo, showing a potential for gene therapy of HCC.