Surgery, chemotherapy and radiotherapy remain as the major treatment strategies for cancers. Some agents such as anti-cancer drugs have capacity to enhance the radiation sensitivity of cancer cells at G2/M phase, leading to an improved radiotherapeutic efficacy. BI6727 is an ATP-competitive pololike kinase 1 (Plk 1)inhibitor and an anti-cancer drug. Using the radio-resistant 9L rat gliosarcoma cells as model, we examined the effect of BI6727 on cell growth and assessed the chemoradiotherapeutic efficiency between 150 kVp conventional irradiation (dose rate of 0.76 Gy min−1 ) and 66 keV synchrotron x-ray broad beam irradiation (dose rate of 46 Gy s−1). Our studies showed that BI6727 significantly caused cell growth arrest at G2/M phase and inhibited 9L cell proliferation with EC50 of 58.1 nM. In combinatory treatment, irradiation of BI6727-treated 9L cells with synchrotron x-rays at a dose rate of 46 Gy s−1 resulted in significant reduction of the cell survival compared to the conventional x-rays at a dose rate of 0.76 Gy min−1. These results indicated that Plk1 inhibitor BI6727 enhanced radio-sensitization of 9L cells in a dose rate dependent manner. For clinical application, irradiation with high dose rate is a promising strategy to improve chemo-radiotherapeutic efficacy for gliosarcoma cancer.