Overexpression of P-glycoprotein (P-gp) is a major cause of multidrug resistance in cancer. P-gp is mainly localized in the plasma membrane and can efflux structurally and chemically unrelated substrates, including anticancer drugs. P-gp is also localized in intracellular compartments, such as endoplasmic reticulum (ER), Golgi, endosomes and lysosomes, and cycles between endosomal compartments and the plasma membrane in a microtubular-actin dependent manner. Intracellular trafficking pathways for P-gp and participation of different Rab proteins depend on cellular polarization and choice of primary culture, cell line or neoplasm. Interruption of P-gp trafficking to the plasma membrane increases intracellular P-gp accumulation and anticancer drug levels, suggesting a potential approach to overcome P-gp-mediated multidrug resistance in cancer.