The rising incidence of ulcerative colitis (UC) in China poses a noticeable health challenge. This study aimed to assess the pivotal role of USP1-associated factor 1 (UAF1) in colitis. UC was induced in male C57BL/6 mice using 2.0% dextran sulfate sodium (DSS). In an in vitro model, RAW264.7 cells were exposed to 200 ng/ml of LPS + ATP. UAF1 expression level was evaluated in colonic tissues, macrophages, and serum samples using quantitative reverse transcription polymerase chain reaction (RT-qPCR). The study assessed weight, disease activity index (DAI) score, myeloperoxidase (MPO) activity, crypt length, inflammatory factors, and epithelial cell function in a mouse model of colitis treated with a UAF1 inhibitor. Microarray analysis identified potential UAF1 targets. Gene interference investigated NLR family pyrin domain containing 3 (NLRP3) involvement in UAF1-induced colitis inflammation. Immunoprecipitation, ubiquitination, and luciferase assays examined the effects of methyltransferase-like 3 (METTL3) methylation on the expression levels of NLRP3 and UAF1. UAF1 expression level was upregulated in colon tissues, RAW264.7 macrophages, and serum samples of colitis mice (P <
0.01). The UAF1 inhibitor (ML-323) enhanced weight and reduced DAI score in colitis mice (P <
0.01). It also decreased MPO activity and ulcer area, and restored crypt length (P <
0.01). UAF1 inhibitor improved epithelial cell function by suppressing NLRP3 activity (P <
0.01). UAF1 promoted inflammation in RAW264.7 macrophages via NLRP3 inflammasome induction (P <
0.01). UAF1 modulated NLRP3 protein expression, leading to reduced NLRP3 ubiquitination induced by LPS + ATP. The m6A-forming enzyme METTL3 enhanced UAF1 stability (P <
0.01) to facilitate UAF1 expression. The findings suggested that METTL3, as an m6A-forming enzyme, could regulate UAF1 mRNA, promoting inflammation in colitis through NLRP3 induction. Inhibiting UAF1 emerges as a potential therapeutic strategy for colitis.