PURPOSE: To investigate the therapeutic effect of exercise preconditioning against acute colitis induced by high-fat diet (HF) plus mild dextran sulfate sodium (DSS) treatment in wild type mice. METHODS: C57BL/6 male mice aged at 6 weeks were assigned to standard chow (SC, n=10) or HF (n=10) or HF plus DSS (HF+DSS, n=10) or exercise preconditioning (EX) with HF+DSS (EX+HF+DSS, n=10), and the mice were subjected to 15 weeks of dietary treatments, with 12 weeks of a moderate treadmill running (50 minutes per session and 5 days per week) and 2 cycles of 5-day DSS (2% w/v) administration included. Measured parameters included clinical symptoms of acute colitis, pro- and anti-inflammatory and chemotactic cytokines, gut barrier proteins, and immunity cells. Gut microbiota was explored by 16S ribosomal RNA amplification sequencing in fecal samples. RESULTS: Chronic exposure to HF resulted in colitis symptoms (significant weight gain, enlargement of the spleen, and shortening of colon length) and histological changes in the colon, decreased gut barrier proteins (zonula occludens-1 and heat shock protein 70), infiltration of immunity cells (neutrophils and monocytes in the colon and blood), increased expression of toll-like receptor 4 in the colon, and increased pro-inflammatory and chemotactic cytokines (interlukin-6, growth-regulated oncogene-α, and monocyte chemoattractant protein-1) and decreased anti-inflammatory cytokine (adiponectin) in the colon and blood, and those pathologic markers of acute colitis were exacerbated by DSS treatment. Exercise preconditioning alleviated the severity of HF+DSS-induced acute colitis and caused symbiotic modifications in gut microbiota, as shown by a lesser abundance of Bacteroides vulgatus (p=0.050) and a greater abundance of Akkermansia muciniphila (p=0.050). CONCLUSIONS: The current findings suggest that exercise preconditioning alleviates the severity of HF+DSS-induced colitis in conjunction with gut microbiota symbiosis in wild-type mice, implying a preventive/therapeutic potential of promotion of physical fitness via regular exercise against this experimentally-induced acute colitis. This study was supported by the National Research Foundation Grant funded by the Korean Government (NRF-2018R1D1A1B07048153 and 2019R1I1A1A01052817).