Objective To determine the effect of ⁶⁰Co γ-ray irradiation on biological behaviors in cultured vascular endothelial cells in vitro. Methods Human umbilical vein endothelial cells (HUVECs) were exposed to ⁶⁰Co γ-ray irradiation at different doses. After irradiation, the proliferation of HUVECs was evaluated by cell growth curve and colony formation assay. Cell cycle was detected by flow cytometry. DNA damage and repair were analyzed by neutral single-cell gel electrophoresis, and the expression of related proteins was detected by Western blotting.Results After γ-ray irradiation, the proliferation of HUVECs was significantly inhibited in a dose-dependent manner. The cells were arrested at G₂/M stage after irradiation exposure. Obvious DNA double-strand breaks and enhanced expression of γH2AX and DNA-PKcs were observed in the HUVECs after irradiation. Conclusion ⁶⁰Co γ-ray irradiation inhibits the proliferation of HUVECs, induces the cells arrested at G₂/M stage, promotes DNA break, and enhances the expression of repair-related proteins in endothelial cells.