Background: Uridine (Urd), which has been reported as a major component of RNA, plays an important role in various biological process including neuroprotection, biochemical modulation and glycolysis, although its role in constipation has yet to be established. Therefore, in this study, we investigated the laxative effects of Urd on chronic constipation., Methods: The constipation phenotypes and their related mechanisms were investigated in the transverse colons of SD rats with loperamide (Lop)-induced constipation after treatment with 100 mg/kg of Urd., Results: The number, weight and water contents of stools were significantly higher in the Lop + Urd treated group than the Lop + Vehicle treated group, while food intake and water consumption of the same group were maintained at a constant level. The thickness of the mucosa layer, muscle and flat luminal surface, as well as the number of goblet cells, paneth cells and lipid droplets were enhanced in the Lop + Urd treated group. Furthermore, the expression of the muscarinic acetylcholine receptors M2 and M3 (mAChR M2 and M3) at the transcriptional and translational level was recovered in the Lop + Urd treated group, while some markers such as Gα and inositol triphosphate (IP3) in their downstream signaling pathway were completely recovered by Urd treatment. Moreover, the ability for mucin secretion and the expression of membrane water channel (aquaporine 8, AQP8) were increased significantly in the Lop + Urd treated group compared with Lop + Vehicle treated group. Finally, the activity of Urd was confirmed in primary smooth muscle of rat intestine cells (pRISMC) based on Gα expression and IP3 concentration., Conclusions: The results of the present study provide the first strong evidence that Urd can be considered an important candidate for improving chronic constipation induced by Lop treatment in animal models.