Background <p>Ulcerative colitis (UC) is an idiopathic chronic intestinal inflammation. It has been reported that macrophages are key cells mediating inflammation. Furthermore, Serpin family B member 5 (SerpinB5) plays a role in the regulation of macrophage phenotype conversion. However, the role and underlying mechanism of SerpinB5 in UC are still unclear.</p> Methods <p>Hematoxylin and eosin staining assay was used to assess colon pathological changes. GO enrichment and KEGG pathway enrichment analyses were conducted to assess the function of the DEGs from RNA-seq data. GSE224758 database, RNA-seq, and GeneCards database were applied to analyze the intersection targets. The mRNA and protein levels of the related genes were determined using RT-qPCR and western blot. The in vitro model of UC was established by stimulating colonic epithelial cells with tumor necrosis factor α (TNF-α). Cell viability, proliferation, and apoptosis were determined using CCK-8, EdU, and flow cytometry. Senescence-associated β-galactosidase (SA-β-Gal) staining and senescence-related proteins p53 and p16 were detected to evaluate the endothelial cell senescence. Inflammatory cytokines and oxidative stress were detected using ELISA and kits. Flow cytometry analysis assessed the percentage of CD11b⁺CD86⁺ and CD11b⁺CD206⁺ cells in mouse-derived macrophages. A mouse model of UC was constructed by injecting C57BL/6 mice with dextran sulfate sodium salt. The interaction of SerpinB5 and F-Box Protein 32 (FBXO32) was confirmed using GST pull-down and coimmunoprecipitation assays. Besides, MeRIP-qPCR, RNA pull-down, and RIP assays were used to examine the molecular mechanism underlying METTL3/m6A/YTHDF1 signaling axis in SerpinB5 expression.</p> Results <p>SerpinB5 expression was increased in UC patients, TNF-α-treated FUC cells, and a mouse model of UC. SerpinB5 silencing repressed TNF-α-induced cell apoptosis, cell senescence, inflammation response, oxidative stress, and M1 macrophage polarization. Meanwhile, SerpinB5 knockdown relieved symptoms of the mouse model of UC and repressed M1 macrophage polarization. SerpinB5 positively mediated the NF-κB pathway by regulating FBXO32. METTL3 improved the stability of SerpinB5 mRNA in an m6A-YTHDF1-dependent manner.</p> Conclusion <p>METTL3/YTHDF1 aggravated UC progression through promoting M1 macrophage polarization via SerpinB5 mRNA m6A modification and FBXO32/NF-κB pathway. These findings indicated that SerpinB5 might be a good and promising therapeutic target for UC treatment.</p>