Abstract: SUMOylation (small ubiquitin-like modification) is a crucial post-translational modification that covalently attaches SUMO (small ubiquitin-like modifier) proteins to lysine residues of substrate proteins, thereby regulating protein stability, subcellular localization, and immune signal transduction. The impact of SUMOylation on the immunogenicity of the SARS-CoV-2 mRNA vaccine-encoded spike (S) protein had not been previously elucidated. Through integrated bioinformatic prediction and experimental validation, this study identified multiple SUMOylation sites on the Omicron variant S protein and confirmed their modification by SUMO proteins in various cell types. Notably, SUMOylation at K421 and K437 residues within the receptor-binding domain was found to play a pivotal role in maintaining vaccine-induced humoral and cellular immune responses, as mutations at these sites significantly reduced serum antibody titers. Furthermore, SUMOylation at the K275 site in the N-terminal domain specifically modulated T cell subset balance by enhancing cytotoxic T cell responses while simultaneously suppressing Th2-type immune reactions. These findings demonstrate that SUMOylation of the S protein expressed by SARS-CoV-2 mRNA vaccines participates in the regulation of antigen-specific immune responses, providing a research foundation for optimizing mRNA vaccine antigen design through SUMOylation-based engineering strategies.