A decrease in the bioavailability of endothelium-derived nitric oxide (NO) is linked to hypercholesterolemia. However, the mechanism by which low density lipoprotein (LDL) mediates endothelial NO synthase (eNOS) dysfunction remains controversial. We investigate the effect of LDL on eNOS regulation in human endothelial cells (ECs). In cultured ECs, a high level of LDL increased the abundance of eNOS and caveolin-1 (Cav-1) in the membrane caveolae and the association of eNOS with Cav-1. Furthermore, it decreased the basal level of NO and blocked NO production stimulated by the calcium ionophore A23187. LDL exposure also increased the formation of stress fibers and the membrane translocation of eNOS. These effects can be blocked by cytochalasin D, an actin cytoskeleton disruptor. In revealing the mechanism underlying the translocation of eNOS, we found that a high level of LDL increased the level of membrane-associated and GTP-formed RhoA and activated the RhoA downstream kinase ROCK-1 activity. Y-27632, a specific inhibitor of ROCK-1, blocked LDL-induced stress fiber formation, eNOS translocation and NO production. In conclusion, a high level of LDL increases the movement of eNOS to membrane caveolae via the increased stress fibers. The RhoA-mediated pathway may play a crucial role in this process in vascular ECs.