Abstract: Constipation is one of the most common functional gastrointestinal disorders, necessitating sustainable and side-effect-free treatment and preventive measures. In this study, four samples with potential constipation-relieving properties-prune juice, comprehensive jiaosu, compounded prebiotics, and compounded probiotics were selected for a 4-week intervention in loperamide-induced constipated mice. Assessments were conducted on constipation indicators, gastrointestinal regulatory neurotransmitters, intestinal barrier systems, and gut microbiota. The results showed that prune juice, compounded prebiotics, and compounded probiotics improved small intestinal propulsion rate, reduced the time to the first black stool, and increased fecal water content. They upregulated the levels of excitatory gastrointestinal regulatory neurotransmitters (motilin (MTL), gastrin (GAS), and substance P (SP) ) in the colon and downregulated the levels of inhibitory gastrointestinal regulatory neurotransmitters (somatostatin (SS) , calcitonin gene-related peptide (CGRP), and nitric oxide). Additionally, they upregulated the expression of tight junction proteins (occludin and claudin mRNA) in the colon, downregulated the relative abundance of Aerococcus, and upregulated the relative abundance of Coriobacteriaceae_UCG-002 and Faecalibaculum. Furthermore, compounded prebiotics and probiotics upregulated the expression of Mucin 2 (MUC2) mRNA in the colon, downregulated the expression of interleukin-1β (IL-1β) mRNA, reduced colonic tissue damage, and further increased the relative abundance of beneficial bacteria such as Ruminococcus_2, Bifidobacterium, Roseburia, and Lactobacillus. In summary, the comprehensive jiaosu used in this study did not alleviate constipation, while prune juice, compounded prebiotics, and compounded probiotics relieved constipation in mice by regulating the relative abundance of key differential bacterial genera, modulating the secretion of gastrointestinal regulatory neurotransmitters, and improving the mechanical barrier of the intestine. Among them, compounded prebiotics and probiotics further regulated the relative abundance of beneficial bacteria, improved the intestinal mucosal and immune barriers, and reduced colonic tissue damage, thereby providing more effective relief from constipation.