This study investigates the role of Sirtuin-3 (SIRT3) in glioblastoma (GBM) and its potential therapeutic effects in inducing ferroptosis. SIRT3 expression was found to be higher in GBM tissues compared to normal brain tissues, and its upregulation was observed during RAS-selective lethal 3 (RSL3)-induced ferroptosis. Inhibiting SIRT3 expression and activity in GBM cells sensitized these cells to RSL3-induced ferroptosis both in vitro and in vivo. Mechanistically, SIRT3 inhibition led to ferrous iron and reactive oxygen species (ROS) accumulation in mitochondria, triggering mitophagy. RNA sequencing analysis revealed that the mitophagy pathway was upregulated, and the cystine transporter SLC7A11 was downregulated upon SIRT3 knockdown. Forced expression of SLC7A11 partially rescued cell viability after RSL3 treatment. Additionally, SIRT3 regulated SLC7A11 transcription through ATF4. Overall, the study provides insights into the novel mechanisms by which SIRT3 protects GBM from ferroptosis and suggests a potential combinatorial approach of targeting SIRT3 and inducing ferroptosis for GBM treatment.This study investigates the role of Sirtuin-3 (SIRT3) in glioblastoma (GBM) and its potential therapeutic effects in inducing ferroptosis. SIRT3 expression was found to be higher in GBM tissues compared to normal brain tissues, and its upregulation was observed during RAS-selective lethal 3 (RSL3)-induced ferroptosis. Inhibiting SIRT3 expression and activity in GBM cells sensitized these cells to RSL3-induced ferroptosis both in vitro and in vivo. Mechanistically, SIRT3 inhibition led to ferrous iron and reactive oxygen species (ROS) accumulation in mitochondria, triggering mitophagy. RNA sequencing analysis revealed that the mitophagy pathway was upregulated, and the cystine transporter SLC7A11 was downregulated upon SIRT3 knockdown. Forced expression of SLC7A11 partially rescued cell viability after RSL3 treatment. Additionally, SIRT3 regulated SLC7A11 transcription through ATF4. Overall, the study provides insights into the novel mechanisms by which SIRT3 protects GBM from ferroptosis and suggests a potential combinatorial approach of targeting SIRT3 and inducing ferroptosis for GBM treatment.