Objective：To investigate the effect of homocysteine （HCY） in promoting senescence of cultured endothelial cells （EC） and the underlying mechanism, Methods： Bovine aortic endothelial cells were isolated by enzymatic digestion method from new born calf and were randomly divided into 4 groups. Cells in control group were not treated with HCY and those in the other 3 groups were treated with HCY with the final concentrations being 0.1, 0.5, and 1.0 mmol/L. The cultured cells were observed morphologically and stained with β-Gal; the cell cycle was examined with flow cytometric method （FCM） and the telomere length was analyzed by Southern blotting. Meanwhile, the contents of nitric oxide（NO）, endothelin （ET）, MDA and the activity of GSH-Px were determined in the supernatants. Results： Compared with control group, the 3 HCY groups had more β-Gal positive cells and shorter telomere length. Cells of G0/G1 phase in the 3 HCY groups were significantly increased than those of control group （P〈0.05） and the cells of S-phase were significantly decreased （P〈0.01）; the contents of NO in supernatants of 3 HCY groups were significantly lower than that of control group（P〈0.05） and the contents of ET, Ox-LDL were markedly higher than that of control group（P〈0.05） ; the contents of MDA and the activity of GSH-Px were similar in all groups. Conclusion： It is indicated that homocysteine may accelerate senescence in endothelial cells, which is not related to oxygenation and reduction.