Pterostilbene, an important analogue of the star molecule resveratrol and a novel compound naturally occurring in blueberries and grapes, exerts a significant neuroprotective effect on cerebral ischemia/reperfusion (I/R), but its mechanism is still unclear. This study aimed to follow the molecular mechanisms behind the potential protective effect of pterostilbene against I/R induced injury. For fulfilment of our aim, we investigated the protective effects of pterostilbene on I/R injury caused by middle cerebral artery occlusion (MCAO) in vivo and oxygen-glucose deprivation (OGD) in vitro. Machine learning models and molecular docking were used for target exploration and validated by western blotting. Pterostilbene significantly reduced the cerebral infarction volume, improved neurological deficits, increased cerebral microcirculation and improved blood-brain barrier (BBB) leakage. Machine learning models confirmed that the stroke target MMP-9 bound to pterostilbene, and molecular docking demonstrated the strong binding activity. We further found that pterostilbene could depolymerize stress fibers and maintain the cytoskeleton by effectively increasing the expression of the non-phosphorylated actin depolymerizing factor (ADF) in the early stage of I/R. In the late stage of I/R, pterostilbene could activate the Wnt pathway and inhibit the expression of MMP-9 to decrease the degradation of the extracellular basement membrane (BM) and increase the expression of junction proteins. In this study, we explored the protective mechanisms of pterostilbene in terms of both endothelial cytoskeleton and extracellular matrix. The early and late protective effects jointly maintain BBB stability and attenuate I/R injury, showing its potential to be a promising drug candidate for the treatment of ischemic stroke.