Ferroptosis, an iron-dependent form of regulated cell death driven by excessive accumulation of lipid peroxides, has become a promising strategy in cancer treatment. Cancer cells exploit antioxidant proteins, including Ferroptosis Suppressor Protein 1 (FSP1), to prevent ferroptosis. In this study, it is found that the E3 ubiquitin ligase TRIM21 bound to FSP1 and mediated its ubiquitination on K322 and K366 residues via K63 linkage, which is essential for its membrane translocation and ferroptosis suppression ability. It is further verified the protective role of the TRIM21-FSP1 axis in RSL3-induced ferroptosis in cancer cells and a subcutaneous tumor model. Moreover, TRIM21 is highly expressed in multiple gastrointestinal (GI) tumors, and its expression is further stimulated upon ferroptosis induction in cancer cells and the KPC mouse model. In summary, This study identifies TRIM21 as a negative regulator of ferroptosis through K63 ubiquitination of FSP1, which can serve as a therapeutic target to enhance the chemosensitivity of tumors based on ferroptosis induction.