Radiation therapy is an integral part of the current therapeutic protocols in colorectal cancer. However, only a small proportion of the patients achieved complete pathological response because of the treatment-induced resistance to radiation. Previous studies have shown that radioresistance is associated with NF-κB activation and that suppression of NF-κB could potentiate the response of colorectal cancer cells to radiotherapy. Icariin, a natural flavonoid, has been shown to suppress NF-κB activity. The present study was carried out to investigate whether icariin could act as a radiosensitizer in colorectal cancer cells and murine model of the colorectal cancer. We also sought to understand the mechanisms underlying the icariin-mediated radiosensitization. Our results showed that icariin enhanced the radiation-mediated anti-proliferative effect both in vitro and in vivo. Further, icariin exerted the anti-proliferative and/or pro-apoptotic effect possibly, by: (1) inducing the cell arrest in G2/M phases of the cell cycle, or by (2) downregulating NF-κB and the anti-apoptotic gene products monitored by this transcription factor. Icariin could also potentiate the efficacy of radiotherapy in the murine model of colorectal cancer. Taken together, these results suggest that the use of icariin may provide with a new approach for sensitizing the radiotherapy in colorectal cancer.