Ape1/Ref-1 is a multifunctional protein with major functions in DNA base excision repair system and redox regulation of several transcription factors. However, overexpression of Ape1/Ref-1 is observed in many types of malignant cancers, leading to point that this protein might be a molecular candidate for anticancer drug treatment. A doxorubicin (DOX) has been widely applied in the chemotherapy of solid tumors including lung. Cytotoxification of DOX is targeting on damage to cellular components, particularly DNA molecules, via generation of reactive oxygen species (ROS). Using specific suppression by short hairpin RNA (shRNA)-based viral vector, an important role of Ape1/Ref-1 on DOX resistance in human lung cancer H1299 cell was investigated in this study. Our data revealed that the stable Ape1/Ref-1 shRNA knockdown had higher susceptibility to DOX compared to the wild type. In accordance, intracellular ROS level was notably accelerated in the Ape1/Ref-1 shRNA defect rather than the wild type. Strikingly, our observation represented that in presence of DOX Ape1/Ref-1 deficiency probably stimulated double strand breaks to DNA, detected by neutral comet assay and gamma-H2AX immunoassay, sensitive methods of genotoxicity. Furthermore, our result showed that DOX-induced apoptosis was also correlated with increase in oxygen radicals-generated damage to DNA. We can conclude that Ape1/Ref1-targeted silencing via shRNA-based interference might be a promising molecular strategy to improve effectiveness of DOX-induced apoptosis in treatment of human lung cancer cells, through induction of oxidative insult. This emphasizes that Ape1/Ref-1 might be a prominent therapeutic target in cancer therapy.