Abstract Slow-transit constipation (STC) is an idiopathic and multifactorial disease, and its pathogenesis is unclear. According to the literature, number or phenotypic abnormality in enterochromaffin cells (ECs) is closely related to the pathogenesis of STC. The Piezo2 protein, an ion channel protein, is specifically expressed in ECs and is crucial for the release of 5-HT and mucus secretion of ECs under stimulation. Previous studies showed that the Piezo2 protein level in the rectal mucosa of STC patients is higher than that in normal rectal mucosa and that Astragaloside IV(AS-IV) promotes Piezo2 protein expression in STC patients. Therefore, this study aimed to elucidate the effect of AS-IV on the intestinal flora of STC mice by constructing STC mouse models and using fecal 16S rDNA sequencing, fecal bacteria transplantation technology, metabonomic sequencing and RNA-Seq to explore the impact of AS-IV on the treatment of STC in vivo and in vitro. The results showed that AS-IV could increase the probiotics levels of Ligilactobacillus, Lachnospiraceae NK4A136, Candidatus Arthromitus and Odoribacter. It can also reduce the level of pathogenic bacteria in Eubacterium fissicatena group. Metabonomic sequencing showed the metabolite 3-bromotyrosine (3-BrY) reduces the caspase-dependent apoptosis of ECs, protects cell survival by inhibiting the activation of the p38 MAPK and ERK signaling pathways induced by loperamide, and promotes the expression of the Piezo2 protein in cells to promote an increase in mucosal secretion and eventually intestinal peristalsis. Keywords Astragaloside IV, enterochromaffin cells, Piezo2, 3-BrY