The blood-brain barrier (BBB) is responsible for maintaining homeostasis of the brain by regulating the chemical environment, immune cell transport, and the entry of toxins and pathogens, and misregulation of the BBB has been shown to be implicated in numerous CNS disorders. One of the challenges in studying the BBB include the bioavailability of therapies, as well as rising multidrug resistance of pathogens. A major contributor to drug resistance and lacking bioavailability of therapies is the over-expression of the broad-specificity P-glycoprotein (P-gp) efflux pump in the endothelial cells of the BBB. However, despite the role of P-gp in affecting the bioavailability, pharmacokinetics and efficacy of therapeutic drugs, the mechanism of the efflux of substrates via P-gp, as well as the inhibition of P-gp activity, are not well understood. To address the mechanisms of P-gp-mediated substrate efflux and P-gp inhibition, we performed multimicrosecond unbiased atomic detail molecular dynamics (MD) simulations of the P-glycoprotein from novel human cryo-EM models as well as X-ray crystal structures in the presence of substrates (doxorubicin, rhodamine-123) and an inhibitor (tariquidar). We compare the differential transport gradient of P-gp substrates and inhibitors between the inward-facing (IF) and outward-facing (OF) conformations of P-gp to propose a model of P-gp turnover. We compare this model of active transport to our model of passive transport across the BBB.