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Breast cancer is the most commonly diagnosed malignancy in women worldwide. Metastatic development is associated with poor prognosis and current therapies provide only limited success. Virotherapy is an emerging strategy for the treatment of cancer that utilizes both replication-competent and genetically modified viruses to selectively kill tumor cells. We have previously shown that Coxsackievirus A21 (CVA21), a wild-type common-cold producing enterovirus, is an effective oncolytic agent against human melanoma xenografts in, vivo. CVA21 specifically targets and lytically infects susceptible cells expressing the CVA21 cellular receptors, intercellular adhesion molecule-1 (ICAM-1) and/or decay-accelerating factor (DAF). Herein, the efficacy of CVA21 as a therapeutic agent against human breast cancer was investigated both in, vitro and in, vivo. Flow cytometric analysis revealed that the human breast cancer cell lines examined expressed significantly elevated levels of surface ICAM-1 and DAF compared to normal breast cell lines, and that all cancerous lines were more susceptible to lytic infection by CVA21 than the normal cells. Through the use of subcutaneous (T47D cells) and orthotopic (MDA-MB-231-luc cells) xenograft SCID mouse models it was demonstrated that a single intravenous injection of CVA21 produced significant regression of pre-established tumors in, vivo, as well as targeting and elimination of metastases in the orthotopic model. Taken together, these findings highlight the exciting potential of CVA21 as a therapeutic agent against both primary and metastatic human breast cancer.