The multidrug transporter P-glycoprotein (P-gp), is pivotal in exporting various chemically dissimilar amphipathic compounds including anti-cancer drugs, thus causing multidrug resistance during cancer treatment. P-gp is composed of two transmembrane domains (TMDs), each containing six homologous transmembrane helices (TMHs). Among these helices, TMH 6 and 12 align oppositely, lining a drug-binding pocket in the transmembrane region which acts as a pathway for drug efflux. Previously, we demonstrated that specific mutations within TMH 6 and 12 resulted in loss of substrate efflux and altered the transport direction from efflux to uptake for some substrates. This suggested the presence of a regulatory switch that governs the direction of transport. In this study, we sought to elucidate the mechanism of switch region modulation of the uptake function by engineering several mutants via substituting specific residues in TMH 6 and 12. We discovered that the alanine substitution of four residues (V974, L975, V977, and F978) within the upper region of TMH 12, along with three residues (V334, F336, and F343) within TMH 6, was sufficient to convert P-gp from an efflux to an uptake pump. Additional mutagenesis of the residues in the middle region of TMH 12 revealed that the uptake function, like efflux, is reversible. Further studies, including molecular dynamics simulations, revealed that the switch region appears to act during the substrate translocation step. We propose that the switch region in TMH 6 and 12, which modulates the direction of transport by P-gp, provides a novel approach to selectively target P-gp-expressing cancer cells.