Background and Purpose: Macrophage infiltration and activation is a critical step during acute colitis. Redox-mediated activation of NLRP3 inflammasomes in macrophages plays a critical role in mediating colonic inflammatory responses. Rhein isolated from the rhizome of rhubarb exhibits anti-inflammatory effects in various diseases. However, its role in regulating acute colonic inflammation is unexplored. Here, we investigated the protective mechanisms of rhein during acute gut inflammation and its regulation of macrophage activation. Experimental Approach: Inhibitory effects of rhein on NLRP3 inflammasomes were evaluated in activated macrophages and a mouse model of colitis. Expression of inflammatory mediators, inflammasome complex and redox-related signalling were analysed by ELISA, Western blots, immunofluorescence staining, and qRT-PCR. The phenotype of macrophages was assessed by flow cytometry. Colonic inflammation was evaluated by histological analysis. Key Results: Rhein significantly decreased IL-1β secretion via NLRP3 inflammasomes by disturbing their assembly in macrophages. Rhein also activated the Nrf2-HO1-NQO1 pathway and inhibited expression of Nox2 subunits and translocation to regulate redox balance. Moreover, rhein attenuated inflammatory responses by mediating macrophage polarization from M1 to M2 phenotype. NF-κB, AP-1, and MAPK signalling were also involved in improving inflammatory conditions by rhein. In mice with acute intestinal inflammation, rhein treatment attenuated clinical features and reduced macrophage infiltration into damaged tissue to alleviate colonic inflammation. Conclusion and Implications: Rhein regulated redox-mediated NLRP3 inflammasome activation to protect against acute colitis, by interfering with macrophage accumulation and polarization. These findings provide a promising strategy of novel compounds for regulating mucosal inflammation in gastrointestinal disorders.