Angiogenesis plays a key role in tumor growth and metastasis. Current antiangiogenic drugs have shown poor clinical benefit making the development of new antiangiogenic agents a clinical need. In this work, we optimized the production of a recombinant fusion protein derived from vasostatin-30 (VS30) and vasoinhibin (Vi), named VS_VI, using Escherichia coli and the central composite experimental design. The bioactivity of soluble VS_VI was compared to VS30, Vi, and angiostatin using in vitro cell cultures and the zebrafish model. Results showed that 21.3 and 17.5 mg/L of total VS_VI and soluble VS_VI, respectively, were attained at pH 7.0 and 28.5 degrees C. In vitro studies showed that purified soluble VS_VI decreased by 2-fold the VEGF-induced HUVEC proliferation compared to VS30 and Vi, showing no effect on 3T3 NIH cell proliferation at 10 nM. In vivo using zebrafish, VS_VI significantly reduced intratumoral angiogenesis at 1 mg/kg compared to a control, VS30, Vi, and angiostatin. VS_VI decreased metastatic dissemination of MDA-MB-468 and MDA-MB-231 cells at 1 mg/kg. Meanwhile, vasoinhibin and angiostatin at 1 mg/kg and vasostatin-30 at 10 mg/kg decreased the dissemination of T47D cells. Results showed VS_VI fusion protein impairs tumor angiogenesis and early dissemination of metastatic breast cancer cells, making it a promising agent in breast cancer treatment.