The development of multidrug resistance (MDR) due to the overexpression of ATP-binding cassette (ABC) transporters remains one of the major obstacles to the success of chemotherapy in clinics. It is of paramount importance to identify novel drug combinations that could inhibit the multidrug efflux of ABC transporters and enhance the chemo-sensitivity of substrate anticancer drugs. In this study, we evaluated avapritinib, a KIT and PDGFRA blocker, for its reversal effects on the drug sensitivity of ABCB1 and ABCG2 overexpressing cells. Our results show that avapritinib significantly enhanced the cytotoxicity of the substrates of both ABCB1 and ABCG2. Mechanistic studies revealed that avapritinib enhances the intracellular accumulation of the substrates of ABCB1 or ABCG2 by directly decreasing their efflux from the cells overexpressing ABCB1 or ABCG2. Moreover, avapritinib did not change the expressional levels or translocation of ABCB1 or ABCG2 protein from the cell membrane to the cytoplasm and stimulates the ATP cleaving activity of both ABCB1 and ABCG2. Taken together, our results open new avenues for the use of avapritinib as cancer chemotherapy, when used in combination with the substrates of ABCB1 or ABCG2.