Abstract: Objective: To investigate the laxative effect and mechanism of galacto-oligosaccharides (GOS) on loperamide (Lop)-induced constipation in mice, which provides novel insights into the treatment of constipation. Methods: Thirty-six male BALB/C mice were randomly divided into three groups, including the blank group, the model group, and the GOS group. The growth status, defecation situation and small intestine transit rate of each group were measured. Subsequently, the morphological changes of the colon tissue were observed using HE staining. The RNA expression levels of aquaporins, mucins, and tight junction proteins in the colon were quantified by real-time fluorescent quantitative polymerase chain reaction. The fecal intestinal microbiota composition was characterized via 16S rRNA gene sequencing, while the concentration of short-chain fatty acids was determined through gas chromatography. Results: Compared to the model group, the GOS group showed a great increase in body weight gain (P<0.05), a notable reduction in the time of the first black stool (P<0.05), and a highly significant elevation in the number of fecal pellets and their moisture content within 5 hours (P<0.05). At the same time, the damage of the colonic tissue was alleviated. The mRNA expressions of AQP-4 and AQP-8 in the colon tissue were reduced by 69.30% and 56.38%, while the mRNA expressions of Muc-2, Muc-3, and ZO-1 were increased by 74.94%, 61.61% and 73.49%, respectively. All these variations were statistically significant (P<0.05). Meanwhile, there was a remarkable increase in the abundance of beneficial bacteria such as Lactobacillus and norank_f_Muribaculaceae in fecal samples. In the end, an obvious rise in the levels of propionic and butyric acids was observed additionally (P<0.05). Conclusion: GOS exhibits a significant effect on alleviating constipation by repairing intestinal barrier dysfunction, regulating intestinal water metabolism, and modulating the composition of gut microbiota potentially.