Enolase-Phosphatase 1 (Enoph1), a newly discovered enzyme of the methionine salvage pathway, has been identified as a candidate gene involved in the modulation of stress responses. In the present study, we investigated the effect of Enoph1 on ischemic blood brain barrier (BBB) injury on an in vitro model of oxygen-glucose deprivation (OGD) and an in vivo rat model of middle cerebral artery occlusion (MCAO). Cell death and apoptosis were assessed by measuring 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide formation, lactate dehydrogenase release and TdT-mediated dUTP-biotin nick end labeling staining. BBB disruption was evaluated based on fluorescein-labeled albumin (FITC-albumin) extravasation. ROS generation was assessed by DCFH staining. Real time-PCR and western blot were used to measure mRNA and protein expression, respectively. CRISPR-activated plasmid and siRNA were applied to manipulate Enoph1 expression in brain microvascular endothelial cells (BMVECs). Exposure of BMVECs to OGD for 6 hours markedly increased Enoph1 expression and decreased the protein levels of its substrate aci-reductone dioxygenase 1 (ADI1) protein, which was accompanied by membrane type 1 matrix metalloproteinase (MT1-MMP) upregulation, increased apoptotic cell death and ROS generation. Increased Enoph1 expression was also observed in ischemic cerebromicrovassels where FITC-albumin extravasation was observed, indicating the co-localization of BBB disruption and increased Enoph1 expression. Knockdown of Enoph1 expression with siRNA significantly inhibited OGD-induce apoptosis, ROS generation, ADI1 protein reduction and MT1-MMP upregulation in BMVECs, while overexpression of Enoph1 CRISPR-activated plasmids exacerbated OGD-induced cell death and ROS generation. Of note, overexpression of ADI1 or knockdown of MT1-MMP with siRNA significantly blocked OGD-induced BMVECs apoptosis. In conclusion, our data demonstrate for the first time that cerebral ischemia activates Enoph1-ADI1-MT1-MMP axis to mediate BMVEC apoptosis and BBB disruption, and Enoph1 may represent a new therapeutic target for ischemic stroke.