This study reveals that fatty acid desaturases 1 and 2 (FADS1/2), which are responsible for polyunsaturated fatty acid (PUFA) biosynthesis, are highly expressed in a subset of triple-negative breast cancer (TNBC) cells with poor prognosis. These cells are more susceptible to ferroptosis, a type of iron-dependent cell death caused by lipid peroxidation. Targeting FADS1/2 by genetic or pharmacological means renders these cells resistant to ferroptosis, while exogenous PUFA supplementation sensitizes them. Inhibition of lipid droplet (LD) formation and turnover reduces the buffering capacity of LD and enhances iron-dependent cell death. These findings were validated in vitro and in vivo in mouse and human-derived models, as well as in a retrospective cohort of TNBC patients. The study highlights the role of lipid metabolism in ferroptosis susceptibility and suggests that targeting FADS1/2 and LD biogenesis could be a promising therapeutic strategy for aggressive TNBC. The results indicate that the PUFA/MUFA ratio is a key determinant of ferroptosis sensitivity, and that altering this ratio can influence the susceptibility of TNBC cells to ferroptosis. The findings provide a new understanding of the metabolic vulnerabilities in TNBC and offer potential therapeutic targets for improving treatment outcomes.This study reveals that fatty acid desaturases 1 and 2 (FADS1/2), which are responsible for polyunsaturated fatty acid (PUFA) biosynthesis, are highly expressed in a subset of triple-negative breast cancer (TNBC) cells with poor prognosis. These cells are more susceptible to ferroptosis, a type of iron-dependent cell death caused by lipid peroxidation. Targeting FADS1/2 by genetic or pharmacological means renders these cells resistant to ferroptosis, while exogenous PUFA supplementation sensitizes them. Inhibition of lipid droplet (LD) formation and turnover reduces the buffering capacity of LD and enhances iron-dependent cell death. These findings were validated in vitro and in vivo in mouse and human-derived models, as well as in a retrospective cohort of TNBC patients. The study highlights the role of lipid metabolism in ferroptosis susceptibility and suggests that targeting FADS1/2 and LD biogenesis could be a promising therapeutic strategy for aggressive TNBC. The results indicate that the PUFA/MUFA ratio is a key determinant of ferroptosis sensitivity, and that altering this ratio can influence the susceptibility of TNBC cells to ferroptosis. The findings provide a new understanding of the metabolic vulnerabilities in TNBC and offer potential therapeutic targets for improving treatment outcomes.