Cancer stem cells have recently been isolated from several different solid tumors. In breast cancer, the CD44(+)CD24(-/low) population is considered to comprise stem-like cells. The identification of cancer stem cells has provided new targets for the development of therapeutics. Oncolytic herpes simplex viruses (oHSVs) are an effective strategy for killing breast cancer cells and treating breast tumors in preclinical models. Here, we examined the efficacy of the oHSV G47 Delta in killing breast cancer stem cells. Human breast cancer cell line SK-BR-3 and human primary breast cancer cells were cultured in suspension under conditions conducive to the growth of stem cells. They generated mammospheres, which had cancer stem cell properties. The proportion of CD44(+)CD24(-/low) cells in these mammospheres exceeded 95%, as determined by flow cytometry. The mammospheres were found to be highly tumorigenic when implanted subcutaneously in nude BALB/c mice. G47 Delta contains the LacZ gene, and X-gal staining of infected cells in vitro and in vivo showed the replication and spread of the virus. G47 Delta was found to be highly cytotoxic to the CD44(+)CD24(-/low) population in vitro, even when injected at low multiplicities of infection, and G47 Delta treatment in vivo significantly inhibited tumor growth compared with mock treatment. This study demonstrates that oHSV is effective against breast cancer stem cells and could be a beneficial strategy for treating breast cancer patients.