Development of resistance to chemotherapy drugs is a significant problem in treating human malignancies in the clinic. Overexpression of ABC transporter proteins, including P-170 glycoprotein (P-gp), and breast cancer resistance protein (BCRP, ABCG2) have been implicated in this multi-drug resistance (MDR). These ABC transporters are ATP-dependent efflux proteins. We have recently shown that nitric oxide (NO) inhibits the ATPase activities of P-gp, resulting in a significant enhancement of drug accumulation and the reversal of multi-drug resistance in NCI/ADR-RES cells, a P-gp-overexpressing human MDR cell line. In this study, we used [O²-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)-piperazin-1 yl]-diazene-1-ium-1-2-diolate] (JS-K), a tumor-specific NO-donor to study the reversal of drug resistance in both P-gp- and BCRP-overexpressing human tumor cells. We report here that while JS-K was extremely effective in reversing adriamycin resistance in the P-gp-overexpressing tumor cells (NCI/ADR-RES); it was significantly resistant to BCRP-overexpressing (MCF-7/MX) tumor cells, suggesting that JS-K may be a substrate for BCRP. Using another NO-donor (DETNO), we show that NO directly inhibits the ATP activities of BCRP, inducing significant increases in the accumulations of both Hoechst 33342 dye and topotecan, substrates for BCRP. Furthermore, NO treatment significantly reversed topotecan and mitoxantrone resistance to MCF-7/MX tumor cells. Molecular docking studies indicated that while DETNO and JS-K bind to ATP binding site in both ABC proteins, binding score was significantly reduced, compared to the ATP binding. Our results indicate that appropriately designed NO donors may find success in reversing multidrug resistance in the clinic.