Background and Purpose Inhibition of NLRP3 inflammasome plays a critical therapeutic potential in the colonic inflammatory responses. The Timosaponin BⅡ (TBⅡ) isolated from the traditional Chinese medicine Anemarrhena asphodeloides has outstanding anti-inflammatory effects in a variety of diseases. Here, we investigated the protective effects of TBⅡ against dextran sulfate sodium (DSS)-induced ulcerative colitis in mice. Experimental Approach Wild-type (WT) and NLRP3 knockout (NLRP3-/-) mice were applied to evaluate the protective effects of TBⅡ in DSS-induced mice colitis. The role of TBⅡ in LPS+ATP-induced cell model was evaluated by inhibiting or overexpressing NLRP3. RNA-seq, ELISA, western blots, immunofluorescence staining and the expression analysis by qPCR were performed to examine the alterations of colonic NLRP3 expression in colon tissues and cells, respectively. Key Results In mice with DSS-induced ulcerative colitis, TBⅡ treatment repaired the intestinal mucosal barrier and alleviated colonic inflammation. RNA-seq analysis and levels of protein expression demonstrated that TBⅡ could prominently inhibit NLRP3 signaling. TBⅡ-mediated NLRP3 inhibition was associated with the alleviation of intestinal permeability and the inflammatory response via blocking the communication between epithelial cells and macrophages. However, pharmacological inhibition of NLRP3 or NLRP3 overexpression significantly impaired TBⅡ-mediated the anti-inflammatory effect. Mechanistically, TBⅡ-mediated NLRP3 inhibition may be partially associated with the suppression of NF-κB. Conclusion and Implications TBⅡ exerted a prominent protective effect against colitis by impeding the crosstalk between epithelial cells and macrophages, partially in the NLRP3-mediated the inhibitory mechanism. These beneficial effects could make TBⅡ a promising drug for relieving colitis.