Melanoma differentiation-associated gene-7 (mda-7), recently classified as interleukin-24 (approved gene symbol IL24), is thought to be a tumor suppressor gene based on the loss of its expression in many different types of cancer. Gene therapy by adenovirus-mediated mda-7 (Ad-mda7) gene transfer has been shown to inhibit the growth of several different tumor cell lines, in vitro and in vivo. We previously demonstrated that Ad-mda7 radiosensitized non-small-cell lung cancer (NSCLC) cell lines by enhancing an apoptosis pathway through the activation of JNK and c-Jun. In the present study, we investigated the efficacy of intratumoral administration of Ad-mda7 combined with ionizing radiation for treating A549 xenograft tumors in nude mice. Substantial and long-lasting inhibition of tumor growth was evident following the combined treatment. Histological examination revealed marked reduction of angiogenic factors (bFGF, VEGF) and microvessel density and enhanced apoptosis in the tumors treated with the combination therapy compared to those treated with Ad-mda7 alone or radiation alone. To confirm the radiosensitizing effect of secreted MDA-7 protein, we performed clonogenic survival assays using human umbilical vein endothelial cells (HUVECs), A549 cells, and normal human lung fibroblasts, CCD16 cells, pretreated with the conditioned medium from 293 cells that had been stably transfected with mda-7 or a control vector. The results showed that MDA-7 protein sensitized HUVECs to ionizing radiation but not A549 cells or CCD16 cells. Our results suggest that Ad-mda7 in combination with radiation enhances apoptosis in the tumors and that secreted MDA-7 protein inhibits angiogenesis by sensitizing endothelial cells to ionizing radiation without affecting other normal cells. We conclude that the combination of mda-7 gene therapy and radiotherapy may be a feasible and effective strategy for treatment of NSCLC.