Purpose: Despite the impressive results with imatinib mesylate (IM) in treatment of chronic myeloid leukemia (CML), the clinical resistance to IM in patients leads to a still serious clinical problems. Autophagy is an adaptation mechanism that is essential for cellular homeostasis in response to various stress environments. Many studies have linked alteration of autophagy with cancer initiation and progression and development of drug resistance. Imatinib inhibits the oncogenic BCR-ABL tyrosine kinase activity and also induce its autophagic proteolysis. However, the role of autophagy in the survival of CML cells remain ill-defined. In this study, we addressed these questions for targeting autophagy to modulate chemoresistance. Methods: To establish IM-resistant sublines, CML K562 cells were maintained and logarithmically growing cells were exposed to increasing concentrations of IM, starting with a concentration of 0.05 μM and increasing gradually by 0.1 μM increments. After the cells acquired the ability to grow in the presence of a specific concentration of IM, the level of resistance was determined. We analyzed the effects of oxidative stress on the cell growth, apoptosis, BCR/ABL expression by western blot analysis, intracellular ROS level, antioxidant enzymes and autophay-associated proteins in both IM-sensitive and IM-resistant K562 CML cells. We further investigated that suppression of autophagy using pharmacological inhibitors (3-MA) enhanced cell dealth induced by IM. Results: Both cells were showed different response each other in degree and pattern by IM exposure, especially in levels of Prx2. Autophagy was downregulated during IM therapy. We demonstrated that suppression of autophagy using either pharmacological inhibitors enhanced cell death induced by IM in cell lines and primary CML cells. IM-resistant K562 CML cells showed profound declines of BCR-ABL levels using autophagy inhibitors with IM. Conclusions: Those results have been implicated autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML. Combining IM use with autophagy inhibition is a promising approach for overcoming drug resistance in Ph+ CML or Ph+ ALL patients especially for IM resistant.