Ferroptosis is a new form of regulated cell death that is dependent on iron- and lipid reactive oxygen species. Emerging evidence indicate that induction of ferroptosis could inhibit the proliferation of diverse cancer cells, which functions as a potent tumor suppressor in cancer. Here, we firstly reported Bufotalin (BT), a natural small molecule, was a novel glutathione peroxidase 4 (GPX4) inhibitor, which could trigger the ferroptosis in non-small cell lung cancer cells. In vitro, BT significantly inhibited the proliferation of A549ácells and induced the ferroptosis, whereas ferroptosis inhibitor or iron chelator significantly reversed the cytotoxicity of BT on A549ácells. Moreover, BT also increased the intracellular Fe²⁺. Subsequently, immunoblotting showed that BT could inhibit the protein expression of GPX4. Notably, BT dramatically accelerated the degradation of GPX4 in A549ácells. Immunoprecipitation assay further certified the increased ubiquitination of GPX4 induced by BT. Nevertheless, BT could not further increase the lipid ROS after silencing of GPX4, suggesting the induction of ferroptosis by BT was dependent on GPX4. Furthermore, BT also observably inhibited tumor growth and promoted lipid peroxidation in vivo. In conclusion, our findings indicated that BT could induce ferroptosis and cause lipid peroxidation by accelerating the degradation of GPX4 and raising the intracellular Fe²⁺, and BT will hopefully serve as a lead compound in developing anti-tumor agents for targeting ferroptosis.