This study evaluated the protective effect of HH2 on beagles orally challenged with enterotoxigenic (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 HH2 significantly reduced the severity of diarrhea after the ETEC challenge ( < 0.05) and increased serum levels of IgG, IgA, and IgM ( < 0.01). HH2 administration also reduced serum levels of DAO at 48 h after the ETEC challenge ( < 0.05), but no significant changes were observed in D-LA ( > 0.05). Oral ETEC challenge significantly reduced the richness and diversity of gut microbiota in beagles not pre-fed with HH2 ( < 0.05), while HH2 feeding and oral ETEC challenge significantly altered the gut microbiota structure of beagles ( < 0.01). Moreover, 14 days of HH2 feeding reduced the relative abundance of - in feces. This study reveals that HH2 alleviates diarrhea caused by ETEC, enhances non-specific immunity, reduces ETEC-induced damage to the intestinal mucosa, and regulates gut microbiota composition.