Aim To investigate the effect of cladribine (2-CdA) on the viability, migration, cell cycle and secretion activity of human umbilical vein endothelial cells, and to evaluate the influence of 2-CdA on cardiovascular system in clinical application. Methods The effect of different concentrations of 2-CdA on the growth of endothelial cells was detected by CCK-8. The cell migration ability was detected by scratch test. The cell cycle was analyzed by flow cytometry. The cell apoptosis was detected by acridine orange and ethidium bromide. The content of nitric oxide (NO) was determined by Gries method. ELISA method was used to detect vascular endothelial growth factor (VEGF) level. Results In the concentration range of 0.4 to 40 mol/L, 2-CdA had an obvious inhibitory effect on endothelial cells, and IC50 was 4.126 mol/L. With the increase of drug concentration and the prolongation of action time, the inhibitory effect of 2-CdA increased. High concentration of 2-CdA for a long time could induce endothelial cell apoptosis. After 5 μmol/L 2-CdA acted on endothelial cells, the migration ability of cells was inhibited, and the cell cycle was significantly blocked in S phase. The staining results of acridine orange and ethidium bromide showed that high concentration of 2-CdA could induce apoptosis of endothelial cells. After the action of 5 μmol/L 2-CdA on endothelial cells, NO and VEGF contents were reduced. Conclusion 2-CdA has the toxic effect of endothelial cells, resulting in cell cycle arrest and cell apoptosis.