Radiotherapy is a commonly used regimen for treating various types of intractable cancers, although the effects depend on the cell cycle of the targeted cancer cell lines, and for irradiation purposes it is therefore critical to establish a protocol for controlling the cell cycle. Here, we showed that a common murine melanoma cell line B16BL6 was more vulnerable to irradiation during the early S phase, and that synchronisation of the cell cycle greatly increased the therapeutic effects of radiotherapy. Cell-sorting experiments, according to cell-cycle phase, using B16BL6 cells demonstrated that cells in the early S phase were the most susceptible to radiotherapy. Gemcitabine, a clinically utilised anti-cancer drug, induced cell-cycle arrest during the early S phase in B16BL6 cells, and thus a synergistic therapeutic effect was observed when irradiation was administered at the right time. Human pancreatic cancer cell line PANC-1 exhibited similar properties to B16BL6 in terms of its radiosensitivity during the S/G2/M phase and also demonstrated a synergistic effect of cell cycle synchronisation. These results show the importance of cell-cycle control in the application of irradiation and suggest a suitable time interval between chemotherapy and radiotherapy, as well as providing useful information for treating intractable cancer.