Unlike the intact fibronectin (FN) molecule, some proteolytic or recombinant fragments of FN possess inhibitory activities on tumor, providing potential strategies in tumor therapeutics. Using the hydrodynamics-based gene delivery technique, we demonstrated that the treatment by in vivo expression of a recombinant CBD-HepII polypeptide of FN, designated as CH50, strongly inhibited the tumor growth, tumor invasion and angiogenesis. Such inhibitory effects of CH50 on tumor were partly ascribed to its influence on the activities of MMP-9 and αvβ3 integrin. The in vivo expressed CH50 decreased both the production and the activity of MMP-9 in tumor tissues. CH50 also down-regulated αvβ3 expression in tumor cells and endothelial cells in vitro. The decreased activity of αvβ3 integrin was proved by its reduced binding ability to fibrinogen and the down-regulation of cdc2 expression. The gene therapy with CH50 not only prolonged the survival of mice bearing hepatocarcinoma in the liver, but also suppressed the growth and invasive ability of tumor in spleen and its metastasis to liver. Taken together, these findings suggest a prospective utility of CH50 in the gene therapy of liver cancer. © 2007 Wiley-Liss, Inc.