One major impediment to central nervous system (CNS) pharmacotherapy is the presence of the drug efflux transporters such as Permeability-glycoprotein (P-gp) at the blood-brain barrier (BBB). These efflux transporters prevent drugs from reaching the brain and exerting their therapeutic effect. In recent years, microRNAs (miRNAs) have shown to be important regulators of P-gp in peripheral organs. Particularly, miR-27a-3p has been shown to play a critical role in the regulation of P-gp in multidrug resistant cancer cells. In brain disorders, altered levels of miR-27a-3p were reported in several diseases associated with alterations in P-gp expression at the BBB. However, effect of altered miR-27a-3p expression on P-gp expression at the BBB remains to be determined. In this study, we investigated the role of miR-27a-3p in the regulation of P-gp expression and activity at the brain endothelium. Levels of miR-27a-3p were modulated by mimic and inhibitor transfection in an in vitro model of human brain endothelial hCMEC/D3 cells. Effect of miR-27a- 3p modulation on P-gp expression and activity was examined and the underlying regulatory mechanisms were explored. Our results showed that transfection of hCMEC/D3 cells with miR- 27a-3p mimic induces expression and activity of P-gp while miR-27a-3p inhibition exerted opposite effects. Mechanistic studies revealed that miR-27a-3p regulates P-gp by mediating Glycogen Synthase Kinase 3 Beta (GSK3ß) inhibition and activating Wnt/ß-catenin signaling. These findings shed light on miR-27a-3p/GSK3ß/ß-catenin as a novel axis that could be exploited to modulate P-gp efflux activity at the brain endothelium and help improving CNS diseases treatment or brain protection.