P-glycoprotein (P-gp) is expressed on brain microvessel endothelial cells of blood-brain barrier (BBB) and elevated after cerebral ischemia. In this study, we explored the influence and potential mechanisms of P-gp on BBB function in experimental ischemic stroke in vivo and in vitro. Middle cerebral artery occlusion/reperfusion (MCAO/R) was created in mice. Oxygen-glucose deprivation/reoxygenation (OGD/R) was performed in brain microvascular vessel-derived endothelial cells (bEnd.3) to mimic ischemia/reperfusion injury in vitro. P-gp-specific siRNA and pharmacological inhibitor cyclosporine A were used to inhibit P-gp, whereas pcDNA3.1 was utilized to overexpress P-gp. Twenty-four hours after reperfusion, acute ischemic stroke outcome, BBB integrity and permeability, autophagic proteins and relative signaling pathways were evaluated. P-gp levels were markedly elevated in mouse brain and endothelial cells following MCAO/R and OGD/R, respectively. P-gp siRNA silencing or pharmacologically inhibiting (cyclosporine A) reduced infarct volume and brain edema, attenuated brain pathology, and improved neurological behavior in association with attenuated accumulation of neutrophils and macrophages, reduced expression levels of inflammatory cytokines (TNF-α and IL-1β), matrix metalloproteinases (MMP-2 and MMP-9) and adhesion molecules (ICAM-1 and VCAM-1). P-gp silence also counteracted BBB leakage, restored the expressions of tight junction proteins (Claudin-5, Occludin and ZO-1), activated autophagic proteins (upregulated LC3-II/LC3-I and Beclin 1, and downregulated P62), and diminished Akt/mTOR signal activity in mice following MCAO/R. In the endothelial cell OGD/R assay, P-gp silence downregulated the expressions of inflammatory cytokines and adhesion molecules, inhibited leukocytes adhesion and migration, increased tight junction protein levels, and activated autophagy, all were reversible by forceful P-gp expression. Additionally, treatment with an autophagy inhibitor (3-methyladenine) abolished protections against ischemic stroke and tight junction proteins reduction followed by P-gp silence. In conclusion, increased P-gp expression after ischemic injury resulted in BBB dysfunction and hyperpermeability by suppressing Akt/mTOR-induced endothelial autophagy.