Ischemic/reperfusions are regarded as the clinical consensus for stroke treatment, which results in secondary injury of brain tissues. Increased blood-brain barrier (BBB) permeability and infiltration of inflammatory cells are responsible for the ischemic/reperfusion injury. In the present study, we aimed to investigate the effects of Agomelatine on brain ischemic/reperfusions injury and the underlying mechanism. MCAO model was established in mice. The expressions of CD68 and claudin-5 in the cerebral cortex were determined using an immunofluorescence assay. Brain permeability was evaluated using Evans blue staining assay. A two-chamber and two-cell trans-well assay was used to detect the migration ability of macrophages through endothelial cells. The expression levels of claudin-5 and MCP-1 in the endothelial cells were determined using qRT-PCR and ELISA. CD68 was found to be up-regulated in the cerebral cortex of MCAO mice but was down-regulated by treatment with Agomelatine. The expression level of down-regulated claudin-5 in the cerebral cortex of MCAO mice was significantly suppressed by Agomelatine. Deeper staining of Evans blue was found in the MCAO group, which was however faded significantly in the Agomelatine treated MCAO mice. The migrated macrophages were significantly increased by hypoxia incubation but were greatly suppressed by the introduction of Agomelatine. The down-regulated claudin-5 by hypoxic incubation in endothelial cells was up-regulated by treatment with Agomelatine. Furthermore, the increased expression of MCP-1 in endothelial cells under hypoxic conditions was significantly inhibited by Agomelatine. Agomelatine prevents macrophage infiltration and brain endothelial cell damage in a stroke mouse model.