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Recently, superparamagnetic nanoparticles of iron oxides have shown great potential in bio-applications, including magnetic resonance imaging contrast enhancement, drug delivery, bio-separation, tissue repair, hyperthermia, and others. In this study, we have explored the enhanced cytotoxicity effect of arsenic trioxide (As2O3) on both sensitive K562 cell line and adriamycin-resistance K562/A02 cell line in the absence and presence of Fe3O4 nanoparticles by MTT assay. The results demonstrate that Fe3O4 nanoparticles could inhibit the function of P-glycoprotein (P-gp) so as to increase the relevant drug accumulation in target cancer cells and thus enhance the cytotoxicity suppression of As2O3. This raises the promising possibility of the great potential application of Fe3O4 nanoparticles in the cancer chemotherapy of leukemia in clinical area.