Iron oxide nanoparticles (IONPs) exhibit remarkable anti-tumoral activity, largely mediated by the generation of reactive oxygen species (ROS). Although certain IONPs can induce ferroptosis, the determinants underlying cell-type-specific sensitivity remain poorly understood. Here, we provide novel mechanistic insight by demonstrating that lysosomal acidity and plasticity critically regulate iron mobilization, ROS compartmentalization, and the ferroptotic response triggered by dimercaptosuccinic acid–coated IONPs (DMSA‑IONPs) in MDA‑MB‑231 breast cancer and U87MG glioma cells. DMSA-IONPs generate ROS in both cell models, however, their subcellular localization markedly differed. In MDA-MB-231 cells, highly acidic lysosomes retained redox-active iron (Fe ^{2 +} ), leading to localized ROS accumulation, lysosomal enlargement and pronounced lipid peroxidation, ultimately inducing ferroptosis, which was reverted by the anti-ferroptosis drug Ferrostatin-1. In contrast, the less acidic lysosomes of U87MG cells released iron into the cytosol and mitochondria, resulting in diffuse ROS production without lipid peroxidation and conferring resistance to ferroptosis despite higher nanoparticle uptake. While IONP‑mediated ROS generation via iron mobilization and Fenton‑like reactions is well established, our findings identify lysosomes as critical determinants of cellular responses to IONPs exposure. Specifically, lysosomal acidity and endolysosomal trafficking govern ferroptotic sensitivity to IONPs by controlling the site of ROS accumulation. Our results demonstrate that IONP-induced ferroptosis depends not only on total ROS levels but also on ROS subcellular distribution, with ROS accumulated in lysosomes triggering lipid peroxidation of these organelles. These insights highlight the importance of evaluating lysosomal physiology across tumor type to optimize nanoparticle-based ferroptosis therapies, particularly for tumors resistant to apoptosis.