Continuous exposure to daunorubicin (DNR) confers resistance against the drug-elicited lethality of leukemic cells and then reduces the remission rate. However, the detailed mechanisms involved in resistance development of leukemic cells to DNR remain unclear. Upregulation of aldo-keto reductases (AKRs) in human leukemic U937 cells was evaluated by gene-specific PCR and western blot analyses, and the contribution of AKRs toward the DNR sensitivity was assessed using gene expression and RNA-interference techniques and specific inhibitors. In addition, DNR reduction and cell differentiation were analyzed by fluorescence high-performance liquid chromatography and flow cytometry, respectively. Treatment with high doses of DNR triggered apoptotic induction of U937 cells through the production of reactive oxygen species (ROS) and a ROS-dependent mechanism. In contrast, DNR, at its sublethal doses, induced the expression of AKR1C1 and AKR1C3, both of which reduced the DNR sensitivity of the cells. The enzymes did not interfere with the cell differentiation caused by DNR, whereas their upregulation facilitated reduction of the anticancer drug and a ROS-derived lipid aldehyde 4-hydroxy-2-nonenal. These results suggest crucial roles of AKR1C1 and AKR1C3 in the acquisition of DNR resistance of leukemic cells by metabolizing both DNR and cytotoxic aldehydes derived from ROS-linked lipid peroxidation.