The oncogenic serine/threonine kinase Pim-1 phosphorylates and activates the ATP-binding cassette transporter breast cancer resistance protein (ABCG2). The ABC transporter P-glycoprotein (Pgp; ABCB1) also contains a Pim-1 phosphorylation consensus sequence, and we hypothesized that Pim-1 also regulates Pgp. Pgp is exported from the endoplasmic reticulum (ER) as a 150-kDa species that is glycosylated to 170-kDa Pgp, translocates to the cell surface, and mediates drug efflux; alternatively, 150-kDa Pgp is cleaved to a 130-kDa proteolytic product by ER proteases or undergoes ubiquitination and proteasomal degradation. Pim-1 and Pgp interaction was studied in GST pull-down and phosphorylation in in vitro kinase assays. Pim-1 knockdown and inhibition effects on Pgp expression were studied by immunoblotting and flow cytometry and on Pgp stability by immunoblotting after cycloheximide treatment. Pim-1 directly interacted with and phosphorylated Pgp in intact cells and in vitro. Pim-1 knockdown or inhibition decreased cellular and cell surface 170-kDa Pgp, in association with both transient increase in 130-kDa Pgp and increased Pgp ubiquitination and proteasomal degradation. Pim-1 inhibition also decreased expression of 150-kDa Pgp in the presence of the glycosylation inhibitor 2-deoxy-d-glucose. Finally, Pim-1 inhibition sensitized Pgp-overexpressing cells to doxorubicin. Thus, Pim-1 regulates Pgp expression by protecting 150-kDa Pgp from proteolytic and proteasomal degradation and enabling Pgp glycosylation and cell surface translocation and thus Pgp-mediated drug efflux. Pim-1 inhibitors are entering clinical trials and may provide a novel approach to abrogating drug resistance.