Overexpression of MDR-1 represents a critical mechanism of drug resistance in cancer. Proteasome inhibitors recently entered the clinic for treatment of multiple myeloma. We provide evidence that the proteasome-inhibitors Bortezomib and MLN273 are both substrates of MDR-1 by using knockdown of MDR-1 via a transposon-based vector system stably expressing siRNA against MDR-1 in MDR-1-overexpressing K562/Dox cells. Notably, the efficacy of MLN273 (EC(50) from 253 ng/ml in MDR-1(+) to 9.7 ng/ml in MDR-1(-) cells) was much more dependent on MDR-1 expression than Bortezomib (EC(50) from 24.9 ng/ml in MDR-1(+) to 4.5 ng/ml in MDR-1(-) cells). Growth inhibition in MDR-1 negative cells was in part due to increased rate of apoptosis. The enhanced inhibitory effect on the proteasome by loss of MDR-1 was corroborated by a reduced proteasomal activity. Our report provides evidence that MLN273 and, to a lesser degree, Bortezomib are both MDR-1-substrates, which might be relevant for drug-resistance in cancer.