ABSTRACTFerroptosis is a form of iron‐dependent regulated necrosis characterized by the abnormal accumulation of peroxidized phospholipids containing polyunsaturated fatty acids (PUFA‐PLs). The conversion of PUFA to PUFA‐CoA is critical for the synthesis of PUFA‐PLs and is reliant on ATP, yet the role of mitochondrial ATP production in regulating ferroptosis remains unclear. In this study, we employed a metabolite deprivation and replenishment system coupled with flow cytometry to investigate the interplay between glutamine metabolism, mitochondrial ATP, and ferroptosis. We demonstrated that depriving cells of glutamine increases intracellular levels of reactive oxygen species (ROS), while also unexpectedly inhibiting ferroptosis induced by cystine deprivation. Mechanistically, glutamine deficiency impaired mitochondrial ATP production, and pharmacological inhibition of mitochondrial ATP export to the cytosol effectively blocked ferroptosis. Further analysis revealed that mitochondrial ATP depletion under glutamine‐deficient conditions hindered the conversion of PUFAs to PUFA‐CoA, thereby limiting PUFA‐PL synthesis and ferroptosis execution. Notably, although glutamine deprivation alone did not directly trigger ferroptosis, it promoted PUFA oxidation and prostaglandin‐endoperoxide synthase 2 (PTGS2) expression via ROS accumulation. Together, our findings highlight the critical role of mitochondrial ATP in ferroptosis regulation and provide new insights into the metabolic control of cell death pathways.