Human reovirus can replicate and induce tumor cell lysis in several cancer types with an activated Ras signaling pathway. The aim of this study was to investigate the oncolytic effect of reovirus against breast cancer cells, and clarify the relationship between the susceptibility to reovirus and HER2 expression, which is associated with the Ras signaling pathway. Reovirus (serotype 3), 6 human breast cancer cell lines and normal mammary gland epithelial cell line were used in this study. The mRNA expression of HER2 receptor was examined by RT-PCR, and the protein of HER2 and activated Ras protein were examined by a Western blot analysis. In vitro, the cytopathic effects, viral protein synthesis, and cell viability on infection by reovirus were examined. Reovirus can infect all the 6 examined cancer cell lines, but the control cell line was not susceptible to the reovirus infection. The cytopathic effect appeared from day 1 after infection, and a 50% or greater cytotolysis was demonstrated at day 7 after infection. The Ras activities in all examined cell lines were higher than those in the control cell line. No relationship was observed in the susceptibility to the reovirus and HER2 expression. The increased Ras activity itself, regardless of the HER2 expression, may therefore play an important role in the susceptibility to reovirus of breast cancer cells. As a result, breast cancer may thus become a candidate target for oncolytic reovirus therapy.