ObjectiveIt was reported fatigue or a high-fat diet triggers diarrhea, and intestinal microbiota may play central roles in diarrhea. Therefore, we investigated the association between the intestinal mucosal microbiota and the intestinal mucosal barrier from fatigue combined with a high-fat diet.MethodThis study divided the Specific pathogen-free (SPF) male mice into the normal group (MCN) and the standing united lard group (MSLD). The MSLD group stood on water environment platform box for 4 h/day for 14 days, and 0.4 mL lard was gavaged from day 8, twice daily for 7 days.ResultAfter 14 days, Mice in the MSLD group showed diarrhea symptoms. The pathological analysis showed structural damage to the small intestine in the MSLD group, with an increasing trend of interleukin-6 (IL-6) and IL-17, and inflammation accompanied by structural damage to the intestine. Fatigue combined with a high-fat diet considerably decreasedLimosilactobacillus vaginalisandLimosilactobacillus reuteri, and among them,Limosilactobacillus reuteripositively associated with Muc2 and negatively with IL-6.ConclusionThe interactions betweenLimosilactobacillus reuteriand intestinal inflammation might be involved in the process of intestinal mucosal barrier impairment in fatigue combined with high-fat diet-induced diarrhea.