Cerebral ischemia-reperfusion injury (CIRI) mainly arises from the clinical treatment of ischemic stroke, induced by the blood-brain barrier (BBB) disruption and infiltrated inflammation. The Sigma-1 receptor (Sigma-1R) is a novel target for neuroprotection, and the α2-receptor agonist pain medication dexmedetomidine displays a neuroprotective effect through activating Sigma-1R. The present study aims to investigate the potential therapeutic effect of dexmedetomidine in a mouse stroke model and hypoxia/reoxygenation(OGD/R)-induced brain endothelial dysfunction. First, we found that Sigma-1R was significantly upregulated in middle cerebral artery occlusion (MCAO) mice by the administration of dexmedetomidine. experiments revealed that dexmedetomidine ameliorated hyperpermeability of the blood-brain barrier (BBB), lowered the expression level of Occludin, and impaired brain function as measured by neurological scores in MCAO mice. assays show that dexmedetomidine alleviated OGD/R-caused cytotoxicity, hyperpermeability, abnormal expression of Occludin, and inflammatory factors in human brain microvascular endothelial cells (HBMVECs). Moreover, blockage of Sigma-1R by its antagonist BD1047 abolished the neuroprotective property of dexmedetomidine in both animal and cell culture experiments. On the basis of these findings, we conclude that dexmedetomidine therapy shows neuroprotection in MCAO mice. Mechanistically, dexmedetomidine alleviated hypoxia/reoxygenation-induced cerebral endothelial dysfunction by activating the Sigma-1R-mediated signaling pathway.