The blood-brain barrier (BBB) maintains a stable brain microenvironment, and is a primary target of ischemic stroke. Breakdown of BBB integrity has been shown to initiate a devastating cascade of events and eventual neuronal loss in cerebral ischemia. Thus, it is important to identify mechanisms by which BBB integrity can be maintained or loss of BBB integrity can be reduced under ischemic stroke conditions. MicroRNAs (miRs) function as a novel class of small noncoding RNAs that negatively modulate protein expression. We and others have recently shown the involvement of miRs in the pathogenesis of ischemic brain injury. However, the functional significance and molecular mechanisms of miRs in regulating cerebrovascular pathology are poorly understood in ischemic stroke. In this study, we investigate the essential role of the miR-15a/16-1 cluster in the regulation of post-ischemic BBB dysfunction. We demonstrated that expression of the miR-15a/16-1 cluster is significantly increased in the cerebral vasculature and cultured BMECs after in vivo and in vitro ischemic stimuli. EC-selective miR-15a/16-1 genetic deficiency leads to reduced BBB leakage and smaller brain infarct in mice after middle cerebral artery occlusion (MCAO). In contrast, EC-selective miR-15a/16-1 transgenic overexpression aggravates BBB damage and brain infarct. Mechanistically, we found that the miR-15a/16-1 cluster can bind to the 3’-UTRs of major BBB tight junctions, Claudins, and inhibit their translation. Genetic manipulation of miR-15a/16-1 level by lentivirus significantly alters the expression of Claudin 2, 5 and 12 in BMECs under the Oxygen Glucose Deprivation (OGD) conditions. Accordingly, EC-selective miR-15a/16-1 transgenic overexpression remarkably inhibits cerebral expression of Claudins in mice following MCAO. These findings suggest that the miR-15a/16-1 cluster functions as a novel master regulator in BBB pathologies and increases brain injury after ischemic stroke. Elucidating the molecular mechanisms of miR-15a/16-1-mediated BBB dysfunction may lead us to discover novel pharmaceutical targets for the development of effective therapies against ischemic stroke.