Vascular endothelial growth factor (VEGF) expression is higher in triple-negative breast cancers (TNBC) compared to other subtypes and is reported to predict incidence of distant metastases and shorter overall survival. We investigated the therapeutic impact of a vaccinia virus (VACV) GLV-1h164 (derived from its parent virus GLV-1h100), encoding a single-chain antibody (scAb) against VEGF (GLAF-2) in an orthotopic TNBC murine model. GLV-1h164 was tested against multiple TNBC cell lines. Viral infectivity, cytotoxicity, and replication were determined. Mammary fat pad tumors were generated in athymic nude mice using MDA-MB-231 cells. Xenografts were treated with GLV-1h164, GLV-1h100, or PBS and followed for tumor growth. Viral infectivity was time- and concentration-dependent. GLV-1h164 killed TNBC cell lines in a dose-dependent fashion with greater than 90% cytotoxicity within 4 days at a multiplicity of infection of 5.0. In vitro, cytotoxicity of GLV-1h164 was identical to GLV-1h100. GLV-1h164 replicated efficiently in all cell lines with an over 400-fold increase in copy numbers from the initial viral dose within 4 days. In vivo, mean tumor volumes after 2 weeks of treatment were 73, 191, and 422 mm(3) (GLV-1h164, GLV-1h100, and PBS, respectively) (p < 0.05). Both in vivo Doppler ultrasonography and immuno-staining showed decreased neo-angiogenesis in GLV-1h164-treated tumors compared to both GLV-1h100 and PBS controls (p < 0.05). This is the first study to demonstrate efficient combination of oncolytic and anti-angiogenic activity of a novel VACV on TNBC xenografts. Our results suggest that GLV-1h164 is a promising therapeutic agent that warrants testing for patients with TNBC.