Breast cancer is a common malignancy among women and is associated with poor 5-year survival rates. Gene radiotherapy, that is, gene therapy combined with radiotherapy, has been extensively studied as a new mode of therapy, but most studies have assessed only one gene. Here, we inserted two anti-oncogenes, ING4 (inhibitor of growth family member 4) and interleukin-24 (IL-24), in the same bicistronic adenovirus vector and explored the effect of dual-gene therapy combined with radiotherapy on breast cancer cells. Flow cytometry assays showed that adenovirus-mediated ING4 and IL-24 expression could suppress growth, promote apoptosis and induce G2/M cell-cycle arrest in MDA-MB-231 cells. Moreover, animal model studies demonstrated that the combination of ING4/IL-24 gene therapy and radiotherapy significantly suppressed cell proliferation and inhibited tumor growth (P<0.05). Mechanistically, the pro-apoptotic response likely involved the upregulation of Bax and Caspase-3 and the downregulation of Bcl-2. Thus, this study indicates that the co-expression of the two anti-oncogenes, ING4 and IL-24, could significantly promote radiotherapy sensitivity in MDA-MB-231 breast cancer cells.