ATP-binding cassette (ABC) transmembrane proteins evidently decrease the intracellular accumulation of substrate chemotherapeutic drugs by extruding them against a concentration gradient, thereby inducing drug resistance. Here we reported the effect of WHI-P154, an irreversible inhibitor of Janus kinase 3 and epidermal growth factor receptor tyrosine kinases, on reversing ABC transporters-mediated drug resistance. We found that WHI-P154 significantly enhanced the sensitivity of ABCG2-overexpressing cells to its substrates. WHI-P154 moderately sensitized ABCB1-overexpressing KB-C2 cells to its substrates whereas showed no sensitizing effect on ABCC1-, ABCC2 or ABCC10-mediated drug resistance. Moreover, WHI-P154 produced a significant increase in the intracellular accumulation of [³H]-mitoxantrone in ABCG2-overexpressing cells. The expression levels nor the localization of the ABCG2 protein was altered after treatment of ABCG2-overexpressing cells with WHI-P154. Further studies indicated that WHI-P154 enhanced the ATPase activity of ABCG2 at low concentrations (<10 μM). Additionally, a docking model predicted the binding conformation of WHI-P154 within the transmembrane region of homology-modeled human ABCG2 transporter. Collectively, these findings highlighted WHI-P154 could significantly reverse ABCG2-mediated multidrug drug resistance by directly blocking the efflux function.