Background <p>Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is characterized by chronic, recurrent, and non-specific intestinal inflammation. This study aimed to elucidate the mechanism of TNIP3 (TNFAIP3-interacting protein 3) in modulating macrophage (Mφ) polarization and intestinal epithelial barrier function in IBD.</p> Methods <p>LPS-induced RAW 264.7 cells and DSS-induced mouse models were used to investigate the role of TNIP3 in IBD. A coculture system of Mφ and MSIE cells was established using Transwell chambers. Mφ polarization was assessed by flow cytometry, ELISA, and Western blotting. The barrier function of MSIE cells was evaluated via transepithelial electrical resistance (TEER) and FITC-dextran permeability assays. NF-κB activation was analyzed by immunofluorescence and Western blotting. The interaction between TNIP3 and TAB1 was examinedipitation (Co-IP).</p> Results <p>TNIP3 overexpression suppressed M1 Mφ polarization and inhibited NF-κB and NLRP3 inflammasome activation. Coculture with TNIP3-overexpression Mφ, enhanced MSIE barrier function, characterized by increased TEER values and reduced membrane permeability was found. The protective effects of TNIP3 overexpression were abolished by the NLRP3 inflammasome agonist BMS-986299 and TAB1 overexpression. TNIP3 alleviated DSS-induced intestinal damage and inflammation, whereas TAB1 overexpression reversed these protective effects in DSS-induced IBD model.</p> Conclusion <p>This study concludes that TNIP3 significantly attenuates inflammatory responses in Mφ by inhibiting M1 Mφ polarization through the TAB1/NF-κB/NLRP3 pathway, and enhances barrier function of IECs in microenvironment. These findings suggest TNIP3 as a potential therapeutic target for IBD treatment.</p> Graphic Abstract <p>Drawn using Biorender online software (<a href="https://app.biorender.com/">https://app.biorender.com/</a>).</p>