To understand the role of intestinal mucosal microbiota on mental stress-related diarrhoea, we collected the intestinal mucosa of mice treated with Folium senna extract gavage combined with restraint and tail pinch stress for 7 days; and intestinal mucosal microbiota characteristics were analyzed by 16S rRNA Pacbio SMRT gene full-length sequencing. The results showed that the diversity (i.e., alpha diversity including the Chao1, Simpson, ACE, and Shannon indices and beta diversity including the NMDS of weighted UniFrac distances) and composition of the microbial community in the intestinal mucosa of mice with diarrhoea and repeated stress changed significantly (P < 0.05). In the co-occurrence network, Staphylococcus sciuri and Escherichia fergusonii was identified as putative keystone species. Moreover, the characteristics of the intestinal microbial species was analyzed by LEfSe, Metastats, and group difference, and ten altered gut microbiota species can be used as characteristic microbes in the mice with diarrhoea and repeated stress: the abundances of Stigmatella aurantiaca, Candidatus arthromitus sp. SFB-mouse, Erythrobacter gaetbuli, Desulfitobacterium hafniense, Ochrobactrum pituitosum, and Candidatus arthromitus sp. SFB-mouse-NL in the model group were significantly lower than those in the control group (P < 0.05); whereas Microbacterium dextranolyticum, Klebsiella pneumoniae, Escherichia sp. BBDP27, and Streptococcus danieliae were enriched in the control group (P < 0.05). Collectively, mental stress-related diarrhoea increased the intestinal microbiota diversity. The species associated with mental stress-related diarrhoea including Microbacterium dextranolyticum, Klebsiella pneumoniae, Escherichia sp. BBDP27, and Streptococcus danieliae were significantly enriched; while the species which are beneficial to mental stress-related diarrhoea are Stigmatella aurantiaca, Candidatus arthromitus sp. SFB-mouse, Erythrobacter gaetbuli, Desulfitobacterium hafniense, Ochrobactrum pituitosum, and Candidatus arthromitus sp. SFB-mouse-NL for its significantly depleted. © King Abdulaziz City for Science and Technology 2020.