Repeated opening of the blood-brain barrier (BBB) after ischemic stroke significantly contributes to brain injury. As the β1 integrin receptor family has been linked to changes in BBB permeability via changes to tight junction (TJ) protein expression and function, we hypothesized that the pro-angiogenic fibronectin receptor α5β1 integrin is acutely upregulated in cerebrovasculature after ischemic stroke and contributes to BBB breakdown via affects on the TJ protein claudin-5. First, α5β1 brain immunohistochemistry after transient middle cerebral artery occlusion (MCAo) in mice demonstrated an increase in core infarct and peri-infarct vascular expression of α5β1 as early as 24 hours after stroke that became even more prominent by post stroke day 2. Next, in vitro studies demonstrated an increase in barrier permeability where stroke was simulated using oxygen-glucose deprivation or TNF-α treatment, as measured by trans-endothelial cell electrical resistance and FITC-dextran permeability, that correlated with an increase in α5β1 expression and changes in claudin-5 expression and localization away from the cell-surface (i.e. away from TJ complexes). Importantly, this increased permeability could be completely prevented by addition of the α5β1 inhibitor ATN-161. Finally, MCAo performed in endothelial cell selective α5β1 knockout mice demonstrated significantly increased post-stroke BBB stability, stable claudin-5 expression, and little to no ischemic injury. Collectively, our results demonstrate that endothelial cell α5β1 integrin expression is increased acutely after stroke, may contribute to BBB breakdown and subsequent expansion of brain injury, and therefore could represent a novel stroke therapeutic target worthy of further investigation.