Breast cancer is one of the most prevalent cancers worldwide. Moreover, despite advances in antineoplastic therapies, induction of tumor cell death without off-target cytotoxicity remains a challenge. However, recent developments in localized radiotherapy and gene therapy have provided an opportunity to explore the potential for these strategies to be additive for the induction of cell death in tumor cells. Here, a novel adenoviral shuttle vector containing the proapoptotic gene Smac under the control of the ionizing radiation (IR)-induced Egr1 promoter was constructed. Following the transient transfection of the construct into MCF-7 and MDA-MB-435 breast cancer cell lines, acute and abundant expression of Smac was observed in response to IR treatment. Further analysis confirmed that the induction of Smac expression resulted in a decrease in cell viability, a slower rate of cell growth, a higher level of apoptosis and altered cell cycle progression. Using a clonogenic assay, IR-induced Smac expression was also found to significantly sensitize Smac-expressing cells to radiation-induced cell death. Taken together, these data suggest that Smac expression driven by the Egr1 promoter has the potential to serve as a radiotherapy-dependent gene therapy agent.