Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have become the standard first-line treatment for advanced lung adenocarcinoma (LUAD) cancer patients with activating EGFR mutations. However, most patients show acquired resistance to EGFR-TKIs, thereby resulting in a modest overall survival benefit. Here, we found that expression level of APE1 was closely associated with TKI resistance in LUAD. Our clinical data show that level of APE1 was inversely correlated with progression-free survival rate and median time to progression in EGFR-mutated LUAD patients. Additionally, we observed increased expression of APE1 in TKI-resistant LUAD cell lines compared to their parental cell lines. Overexpression of APE1-protected TKI-sensitive LUAD cells from TKI-induced cell growth inhibition and cell death. In contrast, inhibition of APE1-enhanced TKI-induced apoptosis, cell growth inhibition and tumor growth inhibition in TKI-resistant LUAD. In addition, we identified that APE1 positively regulates Akt activation and APE1 overexpression-induced TKI resistance was attenuated by inhibition of Akt activity. Finally, we demonstrated that inhibition of the redox function of APE1 enhances the sensitivity of TKI-resistant LUAD cells to TKI treatment and inhibits Akt phosphorylation in TKI-resistant LUAD cells, but not by inhibition of the APE1 DNA repair function. Taken together, our data show that increased expression of APE1 significantly contributes to TKI resistance development in LUAD, and targeting APE1 may reverse acquired resistance of LUAD cells to TKI treatment. Additionally, our data show that APE1 regulates TKI resistance in LUAD cells by activating Akt signaling through a redox-dependent mechanism.