The aim of the present study was to investigate the radiosensitizing effect of genistein, and the corresponding mechanisms of action on breast cancer cells with different estrogen receptor (ER) status. Human breast cancer cell lines such as MCF-7 (ER-positive, harboring wild-type p53) and MDA-MB-231 (ER-negative, harboring mutant p53) were irradiated with X-rays in the presence or absence of genistein. Cell survival, DNA damage and repair, cell cycle distribution, cell apoptosis, expression of proteins related to G₂/M cell cycle checkpoint and apoptosis were measured with colony formation assays, immunohistochemistry, flow cytometry and western blot analysis, respectively. Genistein showed relatively weak toxicity to both cell lines at concentrations in the range of 5-20 μM. Using the dosage of 10 μM genistein, the sensitizer enhancement ratios after exposure to X-rays at a 10% cell survival (IC₁₀) were 1.43 for MCF-7 and 1.36 for MDA-MB-231 cells, respectively. Significantly increased DNA damages, arrested cells at G₂/M phase, decreased homologous recombination repair protein Rad51 foci formation and enhanced apoptotic rates were observed in both cell lines treated by genistein combined with X-rays compared with the irradiation alone. The combined treatment obviously up-regulated the phosphorylation of ATM, Chk2, Cdc25c and Cdc2, leading to permanent G₂/M phase arrest, and up-regulated Bax and p73, down-regulated Bcl-2, finally induced mitochondria-mediated apoptosis in both cell lines. These results suggest that genistein induces G₂/M arrest by the activation of the ATM/Chk2/Cdc25C/Cdc2 checkpoint pathway and ultimately enhances the radiosensitivity of both ER+ and ER- breast cancer cells through a mitochondria-mediated apoptosis pathway.