Human　P-glycoprotein　(P-gp)　belongs　to　ATP-Binding　Cassette　(ABC)　transporter,　which　can　export　varieties　of　anti-cancer　drugs　out　of　tumor　cells,　resulting　in　multidrug　resistance　(MDR)　of　tumor　cells.　We　utilized　targeted　molecular　dynamics　simulation　to　explore　the　allosteric　transition　of　human　P-gp　from　outward-　to　inward-facing　state.　The　results　show　that　NBDs　experience　translational　and　rotational　movement,　caused　by　the　successive　hydrolysis　of　ATP　molecules.　The　reorientation　of　the　TMDs　begins　with　the　closing　of　the　extracellular　gate,　followed　by　the　opening　of　the　cytoplasmic　gate.　The　opening　delay　of　the　cytoplasmic　gate　is　due　to　the　extensive　electrostatic　interactions　here.　The　sequence　conservation　analysis　reveals　that　charged　residues　in　TM3,　4　are　highly　conserved　for　achieving　the　unidirectional　transport　property.　Our　results　give　detailed　movement　and　energy　analyses　to　the　reverse　allosteric　process　of　human　P-gp　and　are　helpful　to　the　understanding　of　the　working　mechanism　of　human　P-gp.