Radioresistance remains a major obstacle for the radiotherapy treatment of cancer. Previous studies have demonstrated that the radioresistance of cancer is due to the existence of intrinsic cancer stem cells (CSCs), which represent a small, but radioresistant cell subpopulation that exist in heterogeneous tumors. By contrast, non-stem cancer cells are considered to be radiosensitive and thus, easy to kill. However, recent studies have revealed that under conditions of radiation-induced stress, theoretically radiosensitive non-stem cancer cells may undergo dedifferentiation subsequently obtaining the phenotypes and functions of CSCs, including high resistance to radiotherapy, which indicates that radiation may directly result in the generation of novel CSCs from non-stem cancer cells. These findings suggest that in addition to intrinsic CSCs, non-stem cancer cells may also contribute to the relapse and metastasis of cancer following transformation into CSCs. This review aims to investigate the radiation-induced generation of CSCs, its association with epithelial-mesenchymal transition and its significance with regard to the radioresistance of cancer.