X-ray-based radiotherapy is one of the most effective therapeutic strategies for breast cancer patients. However, radioresistance and side-effects continue to be the most challenging issues. B-cell translocation gene 1 (BTG1) is a member of the BTG/Tob family, which inhibits cancer growth and promotes apoptosis. We, therefore, hypothesized that BTG1 plays an important role in the radiosensitivity of breast cancer cells. In the present study, breast cancer cell lines that stably overexpressed BTG1 were used to investigate the effects of BTG1 on cell radiosensitivity in vitro. We found that overexpression of BTG1 enhanced the radiosensitivity both of p53-mutant breast cancer MDA-MB-231 cells and p53 wild-type breast cancer MCF-7 cells. We also found that overexpression of BTG1 along with irradiation induced cell cycle G2/M phase arrest, promoted the formation of reactive oxygen species (ROS), increased the rate of chromosomal aberrations and increased cell apoptosis. Further investigation indicated that BTG1 overexpression along with irradiation was involved in inhibition of the PI3K/Akt signaling pathway. Importantly, the finding that BTG1 promoted ionizing radiosensitivity of breast cancer cells in vitro was confirmed in an animal model. Taken together, our data suggest that BTG1 overexpression combined with radiation therapy increases the therapeutic efficacy of breast cancer treatment via regulation of the cell cycle and apoptosis-related signaling pathways.