Brain microvascular endothelial cells (BMECs) dysfunction is related to the pathogenesis of neurovascular complication of diabetes mellitus that adversely lead to various CNS disorders. Mitoquinone (MitoQ) is a mitochondria targeted antioxidant that exerts multiple protective effects in many oxidative damage-related diseases. In this study, we determined the protective effects of MitoQ on high glucose (HG)-induced BMECs injury and investigated the underlying mechanism. We found that HG significantly reduced the expression of Nrf2 and HO-1, decreased mitochondrial membrane potential, increased intracellular and mitochondrial reactive oxygen species (ROS) generation, induced cytoskeletal damage and apoptosis in BMECs. In addition, Mito tempol, a mitochondrial ROS scavenger, significantly reduced HG-induced mitochondrial ROS production and attenuated cytoskeletal damage and cell apoptosis, suggesting MtROS production was involved in HG-induced BMECs injury. Moreover, we found that MitoQ treatment significantly upregulated the expression of Nrf2 and HO-1 in HG-induced BMECs, which is accompanied by improved mitochondrial membrane potential and decreased MtROS production. Meanwhile, MitoQ treatment also remarkably attenuated HG-induced cytoskeletal damage and cell apoptosis in BMECs. However, inhibitor of Nrf2 with ML385 impaired the protective effects of MitoQ in HG-induced BMECs. In conclusion, our results suggest that MitoQ exerts protective effect on HG-induced BMECs injury via activating Nrf2/HO-1 pathway.