Drug resistance is a major obstacle to chemotherapy success in leukaemia. Although ABCB1 (MDR1) overexpression represents a critical mechanism of drug resistance, modulation of ABCB1 shows unsatisfactory clinical outcome. Recent studies showed that MCL1 was upregulated in numerous haematological and solid tumour malignancies. The present study found that patients with newly diagnosed or relapsed/refractory leukaemia expressed higher MCL1 levels than patients that were in complete remission. We demonstrated that overexpression of MCL1 decreased sensitivity of human leukaemia cell lines to cytotoxic drugs and inhibited drug-induced apoptosis. Specific downregulation of MCL1 via RNA interference sensitized multidrug resistant leukaemia cells towards chemotherapy and induced apoptosis. Our study also demonstrated that MCL1 and ABCB1 mediated drug resistance through different mechanisms and the depletion of both MCL1 and ABCB1 showed an additive effect in reversing drug resistance and promoting drug-induced apoptosis. Thus, this study documented an important role of MCL1 in drug resistance and apoptosis. Simultaneous targeting of MCL1 and ABCB1 could be a novel approach to overcome drug resistance in leukaemia.