As an inhibitor of the Notch signaling pathway, N-[N-(3,5-difluorohenacetyl)-l-alanyl]-S-phenylglycine tert-butyl ester (DAPT) may protect brain tissue from serious ischemic injury. This study aimed to explore neuroprotection by DAPT after cerebral ischemia/reperfusion (I/R) injury. DAPT was intraperitoneally injected 3 hours before the establishment of a focal cerebral I/R model in the right middle cerebral artery of obstructed mice. Longa scores were used to assess neurological changes of mice. Nissl staining and TdT-mediated dUTP-biotin nick-end label-ing staining were used to examine neuronal damage and cell apoptosis in the right prefrontal cortex, while immunofluorescence staining was used to detect glial fibrillary acidic protein- and Notch1-positive cells. Protein expression levels of Hes1 and Hes5 were detected by west blot assay in the right prefrontal cortex. Our results demonstrated that DAPT significantly improved neurobehavioral scores and relieved neuronal morphological damage. DAPT decreased the number of glial fibrillary acidic protein- and Notch1-positive cells in the right prefrontal cortex, while also reducing the number of apoptotic cells and decreasing interleukin-6 and tumor necrosis factor-α contents, and simultaneously downregulating Hes1 and Hes5 protein expression. These findings verify that DAPT alleviates pathological lesions and strengthens the anti-in-flammatory response after cerebral I/R injury. Thus, DAPT might be developed as an effective drug for the prevention of cerebral I/R injury.