Eucommia ulmoides leaves (EULs), referred to as Du-zhong, are utilized to lower blood pressure and improve liver and kidney tone, and also have been applied to cardiovascular disease in Korea, China, and Japan. Endothelial dysfunction, which is caused by endothelial nitric oxide synthase (eNOS) uncoupling, is an initial step in atherosclerosis. In this study, we investigated the protective effects of EUL aqueous extract against ox-LDL-induced eNOS uncoupling and its possible mechanisms in human umbilical vein endothelial cells (HUVECs). A EUL component, aucubin, was also applied to ox-LDL-exposed HUVECs. Whereas ox-LDL significantly decreased nitric oxide (NO) levels in HUVECs, EUL extract and aucubin led to significant recovery of NO levels. When treated with ox-LDL in the presence of EUL extracts or aucubin, O ₂ ^- production was markedly reduced in HUVECs compared to treatment with ox-LDL alone. EUL extract and aucubin also led to recovery of phospho-eNOS Thr495 expression, a critical signaling component in eNOS uncoupling, suggesting that EUL has regulatory effects against eNOS uncoupling and might play preventive/regulatory roles against vascular endothelial dysfunction.