The present study was aimed to investigate whether combination of molecular targeting therapy, a dual PI3K/mTOR inhibitor (BEZ235), with radiation can enhance the radiosensitivity of colorectal cancer cells (CRC). K-RAS mutant CRC cells (HCT 116 and SW 620) and wild type CRC cells (HT 29) were irradiated with different dose of radiation (0-6 Gy). The synergistic effects of combining radiation with different concentration of BEZ235 (0-10 nM) pretreatment were demonstrated by cell survival assay. When comparing with radiation alone and BEZ235 alone, the combination of BEZ235 pretreatment and radiation resulted in an increased percentage of sub-G1 phase cells, and an increased number of γ-H2AX/cell (DNA double strand breaks). Radiation up-regulated AKT/mTOR signaling pathway, including the activation of phospho (p)-AKT, p-mTOR, p-eIF4E, and p-rpS6; and this activated AKT/mTOR signaling pathway was attenuated by BEZ235 pretreatment. In addition, BEZ235 blocked double strand break repair induced by radiation through attenuating the activation of ATM and DNA-PKcs and sensitized CRC cells to radiation. In vivo model, the tumor size and the expression pattern of p-mTOR, p-eIF4E, and p-rpS6 were significantly decreased in combined group than radiation alone or BEZ235 alone. Our findings indicate that the administration of BEZ235 before radiation enhances the radiotherapeutic effect of CRC cells both in vitro and in vivo.