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Background Intestinal barrier dysfunction is a central pathological feature of ulcerative colitis (UC), but the epigenetic mechanisms linking metabolism to barrier integrity remain poorly understood. ALOX15 is upregulated in UC, yet its role in histone lactylation—a newly identified glycolysis-derived epigenetic modification—and intestinal barrier regulation has not been explored.Methods ALOX15 expression was assessed in colonic tissues from UC patients, DSS-induced colitis mice, and LPS-treated intestinal epithelial cells (IEC-6 and MCEC). ALOX15 knockout mice and siRNA/CRISPR-Cas9-engineered cell lines were employed to evaluate intestinal barrier function (FD-4 permeability, TEER, tight junction proteins ZO-1, occludin, claudin-1), glycolytic activity (ECAR, glucose uptake, lactate production, glycolytic enzymes HK-2, GLUT1, PKM2, LDHA), and histone lactylation levels (Pan-Kla, H3K18la). The interaction between ALOX15 and HDAC3 was examined by co-immunoprecipitation and immunofluorescence. Pharmacological inhibition of ALOX15 (PD146176) and glycolysis (2-DG, Oxamate) was applied to validate the mechanism.Results ALOX15 was significantly elevated in UC patients and correlated with disease severity. In DSS-induced colitis mice and LPS-treated IEC-6/MCEC cells, ALOX15 upregulation was accompanied by enhanced glycolysis, lactate accumulation, and increased Pan-Kla and H3K18la levels, along with disrupted tight junction proteins and increased FD-4 permeability. ALOX15 deletion or PD146176 treatment restored tight junction expression, reduced FD-4 permeability, suppressed glycolytic enzyme expression, and decreased H3K18la levels. Mechanistically, ALOX15 directly interacted with HDAC3, a key eraser of lactylation. HDAC3 expression was downregulated in UC models, and ALOX15 deficiency restored HDAC3 levels. Co-IP confirmed that ALOX15 was associated with Pan-Kla in an HDAC3-dependent manner. Oxamate, an LDHA inhibitor, phenocopied the protective effects of ALOX15 inhibition, ameliorating DSS-induced colitis and restoring barrier integrity in vivo.Conclusions ALOX15 promotes UC intestinal barrier dysfunction by enhancing glycolysis and suppressing HDAC3, leading to increased H3K18la ( IDDF2026-ABS-0080 Figure 1. Graphical Abstract). Targeting the ALOX15-glycolysis-HDAC3 axis represents a novel epigenetic therapeutic strategy for UC. (Acknowledgements: This study was funded by National Natural Science Foundation of China (82370564))Abstract IDDF2026-ABS-0080 Figure 1