BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory disease linked to early-onset colorectal cancer and metabolic abnormalities. While intestinal lactate disturbances are observed in UC, the role of lactate and lactylation in its pathogenesis remains unclear. The lack of specific biomarkers reflecting these processes limits understanding of their biological significance. METHODS: UC subtypes were classified using ConsensusClusterPlus and NMF based on LRGs. Immune infiltration was assessed with ssGSEA, xCell, and CIBERSORT. WGCNA identified subtype-specific gene modules, and Lasso regression pinpointed hub genes. Single-cell analysis determined cellular localization, while WB and IHC validated findings in clinical, mouse, and cell models. Prognostic machine learning models evaluated the clinical significance of these results. RESULTS: LRGs distinguished UC patients from controls and stratified them into high and low immune infiltration groups. MSN and MAPRE1, strongly linked to UC, showed elevated expression in vitro and in vivo. They aid in diagnosing UC and UC-associated colorectal cancer and serve as predictors of UC severity and response to immunosuppressants. CONCLUSION: Using high-throughput transcriptomic data, we identified hub LRGs and highlighted the role of lactate-mediated lactylation in UC. MSN and MAPRE1 were confirmed to be upregulated in an inflammatory environment, underscoring their potential for personalized UC diagnosis and treatment.