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Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, with temozolomide (TMZ) resistance being a major barrier to improved clinical outcomes. Dysregulated autophagy is closely associated with TMZ resistance in GBM, but the role of sodium/hydrogen exchanger 1 (NHE1)-a key regulator of intracellular pH homeostasis-in this process remains uncharacterized. We established a TMZ-resistant GBM cell line (U251TR) and observed that U251TR cells displayed higher autophagic activity, increased reactive oxygen species (ROS) levels, and reduced apoptotic rates compared to parental U251 cells. Treatment with the NHE1-specific inhibitor HOE-642 dose-dependently suppressed cell viability, inhibited NHE1 activity, reduced intracellular ROS production, and promoted apoptosis in U251TR cells, with a positive feedback loop identified between NHE1 and ROS. Mechanistically, HOE-642 blocked autophagic initiation and flux primarily via the ROS-AKT/ERK signaling axis, in which reduced ROS downregulated p-AKT and p-ERK1/2, where ERK exerted pro-autophagic effects and AKT exerted anti-autophagic effects. In vivo, combined HOE-642 and TMZ treatment significantly reduced xenograft tumor volume and weight, inhibited autophagy, and activated apoptosis compared to TMZ monotherapy, without causing evident systemic toxicity. Our findings identify a novel NHE1-ROS-AKT/ERK-autophagy regulatory axis in TMZ-resistant GBM, validating NHE1 as a potential therapeutic target to enhance TMZ sensitivity by disrupting the autophagy-apoptosis balance, and providing a new strategy for GBM treatment.