In this study, the effect of MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal-H) at a low dose on radiotherapeutic efficacy and its accurate mechanism of radiosensitization were investigated in human non-small cell lung cancer. The effect of MG132 on ionizing radiation (IR)-induced cytotoxicity, cell proliferation and survival of A549 cells was evaluated. The protein expression modulated by MG132 and IR were inspected by Western blot analysis. To determine in vivo radiotherapeutic efficacy, tumor growth delay was analyzed in a A549 tumor-bearing xenograft mouse model after single or repeated treatment of MG132 and/or IR. Induction of apoptosis and change of c-Myc expression in the tumor tissue was explored by histological analysis. MG132 at a non-toxic dose enhanced the radiation-induced cytotoxicity of A549 cells, accompanying a significant decrease of c-Myc expression. Suppression of c-Myc expression by small interfering RNA (siRNA) displayed enhancement of radiosensitivity similarly to MG132 treatment. Tumor growth in the xenograft mice was markedly delayed by systemic administration of MG132 combined with IR. In vivo down-regulation of c-Myc and increased induction of apoptosis were simultaneously observed in the tumor tissues followed by combinational treatment of MG132 and IR. The results reveal a novel mechanism for proteasome inhibitor-mediated radiosensitization in which c-Myc down-regulation is involved.