CH50, a recombinant CBD-HepII polypeptide of human fibronectin, was shown to suppress tumor metastasis in murine hepatocarcinoma and melanoma models. However, the effect of CH50 on human cancer cells is still not clear. Here we evaluated the efficiency of CH50 delivered by recombinant adeno-associated virus (rAAV) vector for breast cancer treatment. Infection of the two human breast cancer cell line MDA-MB-231 and MDA-MB-468 with a rAAV2 vector encoding CH50 resulted in secretion of soluble CH50. In vitro rAAV-CH50 transduction inhibited adhesion to ECM molecules, and transwell migration and invasion of breast cancer cells induced by fibronectin. In both breast cancer cells, rAAV-CH50 targeted αVβ3 signaling, namely inhibited the expression of αVβ3, the activation of FAK, the upregulation of cdc2, and the production and activation of MMP-9 by ECM molecules stimulation. rAAV-mediated tail vein transfusion and stable expression of CH50 in the liver resulted in the long-term presence of CH50 in sera of nude mice. Sustained CH50 expression mediated by rAAV vector suppressed the growth and spontaneous metastasis of orthotopic breast cancer xenograft, experimental metastasis of circulating breast cancer cells, and improved the long-term survival of breast tumor-bearing mice. These findings suggest for the first time that rAAV-CH50 gene therapy may present a novel and promising strategy for treatment against metastatic breast cancer.