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This study evaluated the protective effect of <i>Bacillus subtilis</i> HH2 on beagles orally challenged with enterotoxigenic <i>Escherichia coli</i> (ETEC). We assessed the physiological parameters and the severity of diarrhea, as well as the changes in three serum immunoglobulins (IgG, IgA, and IgM), plasma diamine oxidase (DAO), D-lactate (D-LA), and the fecal microbiome. Feeding <i>B. subtilis</i> HH2 significantly reduced the severity of diarrhea after the ETEC challenge (<i>p</i> < 0.05) and increased serum levels of IgG, IgA, and IgM (<i>p</i> < 0.01). <i>B. subtilis</i> HH2 administration also reduced serum levels of DAO at 48 h after the ETEC challenge (<i>p</i> < 0.05), but no significant changes were observed in D-LA (<i>p</i> > 0.05). Oral ETEC challenge significantly reduced the richness and diversity of gut microbiota in beagles not pre-fed with <i>B. subtilis</i> HH2 (<i>p</i> < 0.05), while <i>B. subtilis</i> HH2 feeding and oral ETEC challenge significantly altered the gut microbiota structure of beagles (<i>p</i> < 0.01). Moreover, 14 days of <i>B. subtilis</i> HH2 feeding reduced the relative abundance of <i>Deinococcus</i>-<i>Thermus</i> in feces. This study reveals that <i>B. subtilis</i> HH2 alleviates diarrhea caused by ETEC, enhances non-specific immunity, reduces ETEC-induced damage to the intestinal mucosa, and regulates gut microbiota composition.