Endothelial dysfunction, which is characterized by damage to the endoplasmic reticulum (ER) and mitochondria, is involved in a variety of cardiovascular disorders. Here, we explored whether mitochondrial damage and ER stress are associated with endothelial dysfunction. We also examined whether and how melatonin protects against oxidized low-density lipoprotein- (ox-LDL-) induced damage in endothelial cells. We found that CHOP, GRP78, and PERK expressions, which are indicative of ER stress, increased significantly in response to ox-LDL treatment. ox-LDL also induced mitochondrial dysfunction as evidenced by decreased mitochondrial membrane potential, increased mitochondrial ROS levels, and downregulation of mitochondrial protective factors. In addition, ox-LDL inhibited antioxidative processes, as evidenced by decreased antioxidative enzyme activity and reduced Nrf2/HO-1 expression. Melatonin clearly reduced ER stress and promoted mitochondrial function and antioxidative processes in the presence of ox-LDL. Molecular investigation revealed that ox-LDL activated the JNK/Mff signaling pathway, and melatonin blocked this effect. These results demonstrate that ox-LDL induces ER stress and mitochondrial dysfunction and activates the JNK/Mff signaling pathway, thereby contributing to endothelial dysfunction. Moreover, melatonin inhibited JNK/Mff signaling and sustained ER homeostasis and mitochondrial function, thereby protecting endothelial cells against ox-LDL-induced damage. © 2021 Peng Li et al.