Clostridium butyricum (CB) can enhance antioxidant capacity and alleviate oxidative damage, but the mechanism by which this occurs remains unclear. This study used enterotoxigenic Escherichia coli (ETEC) K88 as a pathogenic model, and the intestinal microbiota as the starting point to explore the mechanism through which CB alleviates oxidative damage. After pretreatment with CB for 15 d, mice were challenged with ETEC K88 for 24 h. The results suggest that CB pretreatment significantly increased the relative abundance of SCFAs-producing bacteria (Lactobacillus), promoted the growth of epithelial cells, and alleviated the oxidative damage induced by ETEC K88.