Gene modified or cytokine activated mesenchymal stem cells (MSCs) have been used as a treatment in various types of cancer. Moreover, irradiation is usually applied as either a standard primary or adjuvant therapy. Here, we showed that the expression of TNF related apoptosis-inducing ligand (TRAIL) and Dickouf-3 (Dkk-3), the promising anticancer proteins, increased in murine adipose-derived mesenchymal stromal cells (AD-MSCs) following activation with TNF-α, resulting in the induction of apoptosis in cancer cells. Also, anticancer effects of TNF-α activated AD-MSCs were intensified with irradiation. In vivo results showed that TNF-α preactivated AD-MSCs combined with irradiation decreased tumor size and increased survival rate in tumor bearing mice. On the other hands, both TNF-α preactivated AD-MSCs with or without irradiation prevented metastasis in ling and liver, and increased apoptosis in tumor mass. Finally, flowcytometry assay demonstrated that naïve AD-MSCs combined with irradiation but not TNF-α activated MSCs with irradiation increased Treg population in lymph node and spleen. Altogether, obtained results suggest that TNF-α activated MSCs combined with irradiation therapy can serve as new strategy in breast cancer therapy.