PURPOSE: This study aimed to investigate the role of a newly isolated strain L.reuteri LY2-2 in colitis in mice and explored the underlying mechanisms. METHODS L. REUTERI: LY2-2 was orally administered to mice with dextran sulfate sodium (DSS)-induced colitis. 5-Aminosalicylic acid (5-ASA) treatment was used as the drug control. RESULTS: The results showed that the disease severity of colitis mice was significantly alleviated. The intestinal inflammation was restricted by synergistically reducing pro-inflammatory cytokines, inhibiting TLR4-NF-κB signaling, restoring the abnormal immune response, and enhancing intestinal barrier function. Of note, L.reuteri LY2-2 showed great potential in modulating macrophages polarization in colonic tissues. Moreover, the gut dysbiosis was improved. The potentially pro-inflammatory pathogenic bacteria such as Helicobacter and Romboutsia decreased and the probiotics including L.rhamnosus and L.plantarum increased. Interestingly, the above pathological indexes in the L.reuteri LY2-2 group were better than those in the 5-ASA group. CONCLUSION: L.reuteri LY2-2 had a better protective effect on DSS-induced colitis via its anti-inflammatory and microbiota-balancing properties, which supports the potential value of this probiotic against colitis. These results contribute to product development of functional probiotics for colitis and provide valuable insights for their mechanisms of biological function to affect human health status.