Objective This study aims to observe whether Cucurbitacin B (CuB) induces ferroptosis in CAL-27 cells derived from tongue squamous cell carcinoma and explore its possible mechanism. Methods CAL-27 cells were treated with different concentrations of CuB (0, 5, 10, 15, 20, and 30 μmol/L). Cell proliferation activity was detected using the cell counting kit-8 (CCK-8) method, and the median inhibition concentration (IC50) was calculated. Cell clo-ning and wound healing assays were used to detect the effects of different concentrations of CuB on the proliferation and migration of CAL-27 cells. CAL-27 cells were treated with different concentrations of CuB, and the changes in intracellular reactive oxygen species (ROS), Fe2+, GSH, and malondialdehyde (MDA) levels were detected. Western blot (WB) was used to detect the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione peroxidase 4 (GPX4), and solute carrier family 7 member 11 (SLC7A11) proteins. Results CCK-8 results showed that CuB at concentrations of 5, 10, 15, 20, and 30 μmol/L significantly inhibited the proliferation activity of CAL-27 cells, with an IC50 of 13.93 μmol/L (P<0.001). The plate cloning assay demonstrated that CuB inhibited the cloning ability of CAL-27 cells (P<0.01). The wound healing assay revealed that CuB significantly shortened the migration distance of CAL-27 cells and inhibited their migration ability (P<0.01). The results of ROS, GSH, MDA, and Fe2+ measurements indicated that, compared with the control group, the fluorescence intensity of ROS and Fe2+ in CAL-27 cells significantly increased after CuB intervention at different concentrations. Meanwhile, the intracellular GSH content significantly decreased, and the MDA content significantly increased, which induced cellular oxidative damage. These changes could be reversed by the ferroptosis inhibitor Fer-1 (P<0.01). WB results showed that CuB intervention at different concentrations significantly downregulated the expression levels of Nrf2, SLC7A11, and GPX4 proteins (P<0.05). Furthermore, the ferroptosis inhibitor Fer-1 reversed the protein expression of Nrf2, SLC7A11 and GPX4 (P<0.05). Conclusion CuB can induce ferroptosis in CAL-27 cells derived from tongue squamous cell carcinoma, and its mechanism may be related to the ability of CuB to regulate the signaling pathway involving Nrf2, SLC7A11, and GPX4.