Introduction Macrophages play a central role in regulating intestinal inflammation, resolution, and tissue repair. However, the upstream regulators that govern their polarization during colitis remain poorly defined. Methods In this study, we used a dextran sulfate sodium (DSS)-induced colitis model to investigate the role of the scaffold protein AF6 in colitis pathogenesis. Results We identify AF6 as a key regulator that promotes pro-inflammatory macrophage polarization and exacerbates colitis.