〔Abstract〕 ObjectiveTo investigate the role of STAT3 phosphorylation in ferroptosis regulation and its impact on cisplatin resistance mechanisms in human osteosarcoma cells. Methods Human osteosarcoma HOS cells and cisplatin-resistant HOS/DDP cells were treated with cisplatin (0.5, 1, 2, 4, 8, 16, 32 mg/L), the ferroptosis inducer Erastin (Era) (2 μmol/L), and/or the ferroptosis inhibitor Ferrostatin-1 (Fer1) (10 μmol/L). Cell viability and proliferation were assessed using the Cell Counting Kit-8 (CCK-8) and colony formation assays, and cell migration was evaluated via a scratch assay. Reactive oxygen species (ROS), intracellular ferrous iron levels, mitochondrial membrane potential, mitochondrial function, malondialdehyde (MDA) levels, and the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio were measured using commercial kits. The mRNA expression of ferroptosis-related genes was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The protein levels of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), phosphorylated STAT3 (p-STAT3), and total STAT3 were determined by Western blot. Results With cisplatin treatment, HOS cells exhibited decreased cell viability, mitochondrial membrane potential, and GSH/GSSG ratio ( P < 0.01), along with elevated levels of ROS, ferrous ion, and MDA content ( P < 0.01). The protein levels of GPX4 ( P < 0.01), SLC7A11, and p-STAT3 also decreased ( P < 0.05). Coadministration with the ferroptosis inhibitor Ferrostatin-1 (Fer-1) reversed these aforementioned effects ( P < 0.05). In HOS/DDP cells, the mRNA levels of ferroptosis-suppressive genes ( GPX4, FTH1, SLC7A11, and AIFM2) were significantly higher than those in HOS cells ( P < 0.05), whereas the expression of ferroptosis-promoting genes ( ACSL4 and PTGS2) was significantly lower ( P < 0.05). The cisplatin-induced reductions in cell viability and mitochondrial membrane potential, as well as the increases in ROS, ferrous ion, and MDA levels, were less pronounced in HOS/DDP cells than in HOS cells. The SLC7A11 protein level showed no significant change. However, combined treatment with the ferroptosis inducer Erastin (Era) resulted in significant decreases in viability, mitochondrial membrane potential, and the GSH/GSSG ratio in HOS/DDP cells ( P < 0.05). Furthermore, the protein levels of p-STAT3, GPX4, and SLC7A11 were also markedly reduced ( P < 0.05). Conclusion The activation of ferroptosis mediated by p-STAT3 enhances cisplatin sensitivity in HOS/DDP cells.