Osimertinib resistance is regarded as a major obstacle limiting survival benefits for patients undergoing treatment of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). However, the underlying mechanisms of acquired resistance remain unclear. In this study, we report that estrogen receptor β (ERβ) is highly expressed in osimertinib-resistant NSCLC and plays a pivotal role in promoting osimertinib resistance. We further identified ubiquitin-specific protease 7 (USP7) as a critical binding partner that deubiquitinates and upregulates ERβ in NSCLC. ERβ promotes osimertinib resistance by mitigating reactive oxygen species (ROS) accumulation. We found that ERβ mechanistically suppresses peroxiredoxin 3 (PRDX3) SUMOylation and thus confers osimertinib resistance onto NSCLC. Furthermore, we provide evidence showing that depletion of ERβ induces ROS accumulation and reverses osimertinib resistance in NSCLC both in vitro and in vivo. Thus, our results demonstrate that USP7-mediated ERβ stabilization suppresses PRDX3 SUMOylation to mitigate ROS accumulation and promote osimertinib resistance, suggesting that targeting ERβ may be an effective therapeutic strategy to overcome osimertinib resistance in NSCLC.Copyright © 2023. Published by Elsevier B.V.