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Radiation-induced DNA double-strand break (DSB) is the most serious type of DNA damage, which can trigger a series of DNA damage response (DDR) signaling, such as cell cycle arrest, DNA repair, apoptosis, etc. As an important process of DDR, cell cycle arrest is considered to provide sufficient time for DNA damage repair before cell division to prevent the damage DNA delivered to daughter cells. PLK1, CDK1 and CDK4/6 are important kinases of controlling cell cycle progression, and the inhibitors of these kinases can block cell cycle at certain phases. Whether they have radioprotective effect needs to be further explored. In this study, human cervical cancer HeLa cells, human normal breast MCF-10A cells and human umbilical vein epithelial cells (HUVEC) were used to determine the radioprotective effects of PLK1 inhibitors Rigosertib, Volasertib, CDK4/6 inhibitor Palbocilib and CDK1 inhibitor Ro-3306, and the related mechanisms were investigated. Flow cytometry assay demonstrated that Rigosertib, Volasertib, and Ro-3306 blocked cells in G2 phase (P < 0.05, P < 0.01, or P < 0.001), and Palbocilib blocked cells in G1 phase (P < 0.05, P < 0.01, or P < 0.001). Immunofluorescence staining assay demonstrated that Rigosertib, Volasertib, and Ro-330 significantly reduced the number of γH2AX foci after γ-ray irradiation (P < 0.001), suggesting that the above agents could promote DNA repair efficiency. HR and NHEJ reporter assays confirmed that Rigosertib and Ro-3306 promoted DNA repair via both pathways of DSBs repair simultaneously efficiency (P < 0.05 or P < 0.001), and Palbocilib and Volasertib could promote HR efficiency. From the above results, the PLK1 inhibitor Rigosertib was preferentially selected for further study. It was found that Rigosertib significantly reduced radiation-induced apoptosis (P < 0.05 or P < 0.01). CCK-8 and colony formation assays demonstrated that Rigosertib promoted the proliferation and viability of irradiated cells (P < 0.05, P < 0.01 or P < 0.001). In summary, we analyzed and compared the effects of four agents, Rigosertib, Volasertib, Palbocilib and Ro-3306, on the radiosensitivity of cells, among which Rigosertib protects the cells from radiation damage most efficiently. We provide a new strategy and experimental bases for the development of radiation protection drugs. [ABSTRACT FROM AUTHOR]