This study investigates the role of SIRT3 in glioblastoma (GBM) cells and its potential to sensitize GBM to ferroptosis. SIRT3, a mitochondrial deacetylase, was found to be highly expressed in GBM tissues compared to normal brain tissues. SIRT3 expression was upregulated during RSL3-induced ferroptosis in GBM cells. Inhibiting SIRT3 expression and activity in GBM cells increased their sensitivity to RSL3-induced ferroptosis both in vitro and in vivo. Mechanistically, SIRT3 inhibition led to the accumulation of ferrous iron and reactive oxygen species (ROS) in mitochondria, triggering mitophagy. RNA sequencing revealed that SIRT3 knockdown upregulated the mitophagy pathway and downregulated SLC7A11, a key regulator of ferroptosis by facilitating cystine import for glutathione (GSH) synthesis. Forced expression of SLC7A11 in SIRT3-knockdown GBM cells restored cystine uptake and GSH levels, partially rescuing cell viability upon RSL3 treatment. SIRT3 regulated SLC7A11 transcription through ATF4. Overall, the study elucidates that SIRT3 protects GBM cells from ferroptosis by inhibiting mitophagy and promoting SLC7A11 expression. Targeting SIRT3 in combination with ferroptosis induction may offer a novel therapeutic approach for GBM treatment. The findings suggest that SIRT3 inhibition sensitizes GBM cells to ferroptosis by promoting mitophagy and reducing SLC7A11 expression, providing insights into a potential combinatorial therapy for GBM.This study investigates the role of SIRT3 in glioblastoma (GBM) cells and its potential to sensitize GBM to ferroptosis. SIRT3, a mitochondrial deacetylase, was found to be highly expressed in GBM tissues compared to normal brain tissues. SIRT3 expression was upregulated during RSL3-induced ferroptosis in GBM cells. Inhibiting SIRT3 expression and activity in GBM cells increased their sensitivity to RSL3-induced ferroptosis both in vitro and in vivo. Mechanistically, SIRT3 inhibition led to the accumulation of ferrous iron and reactive oxygen species (ROS) in mitochondria, triggering mitophagy. RNA sequencing revealed that SIRT3 knockdown upregulated the mitophagy pathway and downregulated SLC7A11, a key regulator of ferroptosis by facilitating cystine import for glutathione (GSH) synthesis. Forced expression of SLC7A11 in SIRT3-knockdown GBM cells restored cystine uptake and GSH levels, partially rescuing cell viability upon RSL3 treatment. SIRT3 regulated SLC7A11 transcription through ATF4. Overall, the study elucidates that SIRT3 protects GBM cells from ferroptosis by inhibiting mitophagy and promoting SLC7A11 expression. Targeting SIRT3 in combination with ferroptosis induction may offer a novel therapeutic approach for GBM treatment. The findings suggest that SIRT3 inhibition sensitizes GBM cells to ferroptosis by promoting mitophagy and reducing SLC7A11 expression, providing insights into a potential combinatorial therapy for GBM.