Quercetin has been reported to inhibit the growth of oral squamous cell carcinoma (OSCC), but the mechanism remains unclear. Therefore, our study aimed to investigate the involvement of sirtuin 3 (SIRT3) and the autophagy-dependent form of cell death, ferroptosis, in the pathogenesis of OSCC, and observe the impacts of quercetin on ferroptosis and SIRT3/AMPK/mTOR-mediated autophagy.SIRT3 knock out or overexpressing SCC15 cell line was generated, treated with indicated drugs, and malondialdehyde (MDA) and ROS levels were measured. Roles of SIRT3 in regulating autophagy-mediated ferroptosis were assessed by immunoprecipitation and Western blotting.SIRT3 overexpression increased levels of MDA and ROS, reducing cell viability, and SIRT3 knockout produced the opposing effect. SIRT3 overexpression upregulated ATG16L1 expression and the conversion of LC3-Ⅰ to LC3-Ⅱ, triggering autophagy. Suppression of autophagy by ATG16L1 knockout impaired SIRT3-triggered ferroptosis. Use of an AMPK inhibitor antagonized the induction of ferroptosis by SIRT3 in SCC15 cells, indicating the involvement of the AMPK/mTOR pathway. Additionally, quercetin significantly increased the levels of SIRT3, p-AMPK, ATG16L1, and the ratio of LC3-Ⅱ/Ⅰ, but reduced cell viability and p-mTOR in SCC15 cells. Autophagy and AMPK inhibitors, or SIRT3 deletion significantly antagonized the impacts of quercetin on the autophagy-mediated ferroptosis in cancer cells.SIRT3 overexpression activated the AMPK/mTOR pathway and triggered ATG16L1-mediated autophagy, promoting ferroptosis in SCC15 cells, and we proposed that quercetin may be a promising therapeutic drug for OSCC.