Salidroside is a major constituent of Rhodiola rosea L. that elicits beneficial effects for ischemic cardiovascular diseases. The aim of this study was to investigate the protective effects of salidroside on endothelial cells apoptosis induced by the hypoxia mimicking agent, cobalt chloride. After challenge with cobalt chloride for 24 h, loss of cell viability and excessive apoptotic cell death were observed in EA.hy926 endothelial cells, and the level of intracellular reactive oxygen species (ROS) increased concentration-dependently. However, the endothelial cell apoptosis and excessive ROS generation were attenuated markedly by salidroside pretreatment. In addition, salidroside inhibited activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP) induced by cobalt chloride, decreased expression of Bax and rescued the balance of pro- and anti-apoptotic proteins. These findings suggest that salidroside protects endothelial cells from cobalt chloride-induced apoptosis as an antioxidant and by regulating Bcl-2 family. Salidroside may represent a novel therapeutic agent for the treatment and prevention of hypoxia and oxidative stress-related diseases.