This study investigated whether radiation-induced overexpression of superoxide dismutase 2 (SOD2) exerts radio-sensitizing effects on tumor cells while having radioprotective effects on normal cells during radio-activated gene therapy for human colorectal cancer. A chimeric promoter, C9BC, was generated by directly linking nine tandem CArG boxes to a CMV basic promoter, after which lentiviral vectors containing GFP and SOD2 gene driven by the C9BC promoter were constructed. Stably transfected HT-29 colorectal cancer cells and CCD 841 CoN normal colorectal cells were irradiated to a dose of 6-Gy, and cell proliferation and apoptosis were observed. Tumor xenografts and peritumoral skin tissue in BALB/c mice were infected with the therapeutic lentivirus and subsequently irradiated with a total dose of 6 Gy. In vitro experiments revealed that radiation-induced SOD2 overexpression inhibited tumor cell proliferation (61.89% vs. 40.17%, P < 0.01) and decreased apoptosis among normal cells (14.8% vs. 9.6%, P = 0.02) as compared to untransfected cells. Similar effects were observed in vivo. Thus radiation-induced SOD2 overexpression via the chimeric C9BC promoter increased the radiosensitivity of HT-29 human colorectal cancer cells and concurrently protected normal CCD 841 CoN colorectal cells from radiation damage.