Growing evidence has demonstrated that fatigue and a high-fat diet trigger diarrhea, and intestinal microbiota disorder interact with diarrhea. However, the association of intestinal mucosal microbiota with fatigue and high-fat diet trigger diarrhea remains unclear. The specific pathogen-free Kunming male mice were randomly divided into the normal group (MCN), standing group (MSD), lard group (MLD), and standing united lard group (MSLD). Mice in the MSD and MSLD groups stood on the multiple-platform apparatus for four h/d for fourteen consecutive days. From the eighth day, mice in the MLD and MSLD groups were intragastric lard, 0.4 mL/each, twice a day for seven days. Subsequently, we analyzed the characteristics and interaction relationship of intestinal mucosal microbiota, interleukin-6 (IL-6), interleukin-17 (IL-17), malondialdehyde (MDA), superoxide dismutase (SOD), and secretory immunoglobulin A (sIgA). Results showed that mice in the MSLD group had an increased number of bowel movements. Compared with the MCN group, the contents of IL-17, and IL-6 were higher ( > 0.05), and the content of sIgA was lower in the MSLD group ( > 0.05). MDA and SOD increased in MLD and MSLD groups. and were the characteristic bacteria of the MSLD group. And were positively correlated with IL-6, IL-17, and SOD. In conclusion, the interactions between , and intestinal inflammation, and immunity might be involved in fatigue and high-fat diet-induced diarrhea.