Significance The multidrug transporter P-glycoprotein protects tissues from xenobiotics and other toxic compounds by pumping them out of cells. This transporter has been implicated in altering the bioavailability of chemotherapeutic drugs and in the development of multidrug resistance in tumor cells. Despite decades of research, the modulation of P-glycoprotein to overcome drug resistance in the clinic has not been successful. Here, by substituting a group of 14 conserved residues in homologous transmembrane helices 6 and 12 with alanine, we generated a mutant that exhibits a change in the direction of transport from export to import for certain drug substrates including the taxol derivative flutax-1. The ability to convert P-glycoprotein into a drug importer provides a strategy to combat cancer drug resistance.