This study aims to investigate the regulation effects of Xuanhuang Runtong tablets (XHRTs) on intestinal microbes and inflammatory signal toll receptor 5 (TLR5)/interleukin-17A (IL-17A) in STC mice. First, high-performance liquid chromatography (HPLC) was used to verify the composition of XHRT and quality control. Then, the defecation ability of STC mice was evaluated by measuring fecal water content and intestinal transit function. The pathological examination of colonic mucosa was observed by Alcian Blue and periodic acid Schiff (AB-PAS) staining. 16S ribosomal DNA (16S rDNA) genes were sequenced to detect the fecal microbiota. Western blotting, immunofluorescence, and real-time fluorescence quantitative PCR (qRT-PCR) were applied to detect the expression of aquaporin 3 (AQP3), connexin 43 (Cx43), TLR5, and IL-17A. The defecation function of the STC mice was significantly decreased. The amount of mucus secretion and the thickness of the colonic mucus layer were decreased, and the number of microbial species in the intestinal wall, such as Firmicutes/Bacteroidetes, anaerobic bacteria, and Alistipes, were also decreased. In addition, the expression of AQP3 and Cx43 was disordered, and the inflammatory factorsTLR5 and IL-17A were activated in the colon. The changes in the above indicators were significantly reversed by XHRT. This study demonstrates that XHRT provides a new strategy for the treatment of slow transit constipation by regulating the activation of the intestinal inflammatory signal TLR5/IL-17A mediated by gut microbes.