P-glycoprotein (Pgp) pumps structurally different substrates out of cells, which leads to decreased intracellular drug concentration. Taxane drugs represent a class of anticancer drugs that induce cancer cell death by stabilizing microtubules. The efficacy of paclitaxel and docetaxel has been limited by multidrug resistance. Cabazitaxel was observed to overcome the drug resistance induced by docetaxel, suggesting differential transport dynamics of the three taxane drugs. Nevertheless, few studies have delved into their underlying drug transport mechanisms. To fill this gap in knowledge, we examined the drug-Pgp interactions using a combination of in vitro and in silico approaches.We first compared ATPase activities of Pgp in the presence of three taxane drugs, and the results revealed distinctive patterns among them. Paclitaxel and docetaxel both stimulated ATP hydrolysis. Cabazitaxel increased ATP hydrolysis at low concentrations but inhibited ATP hydrolysis at high concentrations. The binding affinity of paclitaxel to Pgp was higher than that of docetaxel and cabazitaxel. Paclitaxel induced different Pgp conformations depending on its concentration: a wide inward-facing open conformation at a high concentration but a slightly occluded conformation at a low concentration. Both docetaxel and cabazitaxel shifted Pgp towards an occluded conformation. Two computational methods, molecular docking and molecular dynamics (MD) simulation, were employed to resolve the fundamental properties of the binding between taxane drugs and Pgp. The result of induced fit docking showed a higher binding affinity of cabazitaxel to the “access tunnel” of Pgp compared to the central binding pocket. This binding preference of cabazitaxel explained its inhibition effect on ATP hydrolysis. MD simulations uncovered protein dynamics showing that cabazitaxel formed more hydrogen bonds with Pgp than docetaxel and paclitaxel, while paclitaxel formed π-π interactions with Pgp. Furthermore, we constructed a proteoliposome-based model to study drug-Pgp interactions in brain lipid environments. Using this model, we observed increased Pgp-mediated ATPase activity in the E. coli lipid environment compared to that in the brain lipid environment. Our research provides the basis for a new generation of anticancer chemotherapy.