Abstract: Objective To study the mechanism of homocysteine（Hcy）at different concentrations and action times on eNOS activity in cultured human umbilical vein endothelial cells（HUVECs）, both in base and calcium ionophore activated state. METHODS HUVECs were collected and cultured to the second passage.Then the second passage of HUVECs were cultured with different concentrations Hcy and in different action times. The expression of HSP90,Akt,Caveolin-1,eNOS,P-eNOS proteins were detected by western blot and the expression of Caveolin-1,eNOS,DDAH mRNA by RT-PCR.RESULTS (1) Compared with control group, both in base and calcium ionophore activated state. The expression of Caveolin-1 group increased obviously(p＜0.05) in concentration and time-dependent manner but the expression of Akt showed a decrease(p＜0.05). The expression of eNOS did not change significantly(p﹥0.05);(2) There was no difference in the expression of HSP90,eNOS,P-eNOS protein and mRNA among every group. DDAH mRNA among every group too..CONCLUSIONS There was no relationship between decreasing activity of eNOS induced by Hcy and eNOS expression, Hcy probably can increase the association of Caveolin-1and eNOS by enhancing the expression of Caveolin-1,and reduce the complex formation of Caveolin-1, Akt,Caveolin-1 in HUVEC both in base and activated state, which can decrease the activity of eNOS.