The activation of inflammasomes (NLRP3 and NLRP1) is central to the pathogenesis of inflammatory bowel disease (IBD). Here we examined the protective effects of a thioredoxin-mimetic peptide CB13 (TXM-CB13), known for its antioxidative stress and anti-inflammatory properties. We examined the effects of TXM-CB13 on dextran sulfate sodium (DSS)-induced colitis and lipopolysaccharide (LPS)-induced NLRP3 inflammasome activation in RAW264.7 macrophages. TXM-CB13 appeared to alleviate symptoms of DSS-induced colitis and to significantly suppress the protein and mRNA levels of NLRP3, Mlck, and IL-1β in colonic tissues. Additionally, TXM-CB13 treatment increased the levels of the intestinal barrier proteins Occludin, ZO-1, and NLRP1, as shown through immunohistochemistry and Western blot analysis. In vitro, TXM-CB13 inhibited LPS-induced TLR4 signaling, reducing MyD88 levels and consequently attenuating the activation of the NF-κB pathways, including p-IκB-α/IκB-α and p-NF-κB-p65/NF-κB-p65. This inhibition further reduced the activation of the NLRP3 inflammasome components, NLRP3, ASC, Caspase-1, GSDMD, and IL-1β. In addition, TXM-CB13 prevented the ROS-mediated dissociation of TXNIP from TRX, inhibiting NLRP3 activation. These findings suggest that TXM-CB13 is a potential therapeutic candidate for IBD through its modulation of the TLR4/MyD88/NF-κB/NLRP3 and ROS/TXNIP/TRX/NLRP3 pathways.