The diarrheal disease causes high mortality, especially in children and young animals. The gut microbiome is strongly associated with diarrheal disease, and some specific strains of bacteria have demonstrated antidiarrheal effects. However, the antidiarrheal mechanisms of probiotic strains have not been elucidated. Here, we used neonatal piglets as a translational model and found that gut microbiota dysbiosis observed in diarrheal piglets was mainly characterized by a deficiency ofLactobacillus, an abundance ofEscherichia coli, and enriched lipopolysaccharide biosynthesis.Limosilactobacillus mucosaeandLimosilactobacillus reuteriwere a signature bacterium that differentiated healthy and diarrheal piglets. Germ-free (GF) mice transplanted with fecal microbiota from diarrheal piglets reproduced diarrheal disease symptoms. Administration ofLimosilactobacillus mucosaebut notLimosilactobacillus reuterialleviated diarrheal disease symptoms induced by fecal microbiota of diarrheal piglets and by ETEC K88 challenge. Notably,Limosilactobacillus mucosae-derived extracellular vesicles alleviated diarrheal disease symptoms caused by ETEC K88 by regulating macrophage phenotypes. Macrophage elimination experiments demonstrated that the extracellular vesicles alleviated diarrheal disease symptoms in a macrophage-dependent manner. Our findings provide insights into the pathogenesis of diarrheal disease from the perspective of intestinal microbiota and the development of probiotic-based antidiarrheal therapeutic strategies.