Background: Although constipation is a common syndrome and a worldwide health problem, the mechanism of underlying remission remains poorly understood. Patients with constipation are becoming younger with a 29.6% overall prevalence in the children, which has captured great attention of scientists because of its epigenetic rejuvenation and recurrent episodes. Despite the usage of rhubarb torelieveconstipation, novel targets and genes involved in target-relevant pathways with remarkable functionalities should still be sought after. Materials and methods: We established a reliable constipation model in C57B/6N male mice using intragastric administration diphenoxylate and the eligible subjects received rhubarb extraction at a dose of 600mg/25g for three days to ameliorate constipation. Resultant constipation was morphological and genetically compared to the different group’s specimen. Results: The constipation mice exhibited thicker muscle layers, improved content of cytokines, including IL-17 and IL-23, and lower content of IL-22. Afterward, the bacterial abundance and diversity varied tremendously. Additionally, SCFA and MLCFA were significantly influenced by the constipation condition accompanied by the enhanced expressions of SCFA receptors, GPR41 and GPR43. Nevertheless, the alterations were reversed to the normal level when constipation mice were treated with rhubarb. Conclusion: This thesis has provided a deeper insight into the confirmation that rhubarb promoted the flexibility of collagen fiber, reduced pro-inflammatory cytokines and enhanced anti-inflammatory cytokines to relieve inflammation, improved intestinal barrier function and maintained intestinal microflora balance, with potential effects on intestinal microenvironment such as affecting the metabolism of fatty acids and polyamines.