Abstract: Objective: Constipation is a common functional gastrointestinal disorder, necessitating sustainable and side-effect-free relief methods. Methods: This study investigated the efficacy of combined prebiotics in a loperamide-induced constipation mouse model. Further assessments included gut microbiota composition, fecal short-chain fatty acid levels, intestinal barrier-related markers, colonic tissue damage, and gastrointestinal regulatory neurotransmitters. Results: Combined prebiotics alleviated constipation by downregulating harmful bacteria (Bilophila, Anaerovorax) and upregulating beneficial bacteria (Ileibacterium, Bifidobacterium), thereby increasing excitatory gastrointestinal neurotransmitters (upregulating gastrin and substance P), improving the intestinal barrier (upregulating mucin-2, occludin, and stem cell factor mRNA expression while downregulating aquaporin-3 mRNA expression), repairing colonic tissue damage, and elevating fecal acetate and butyrate levels. These changes led to a 27% reduction in time to first black stool excretion and 46% increase in fecal water content compared to the model group. When combined with prune juice, the prebiotics formulation further enhanced the abundance of beneficial bacteria (Odoribacter, Faecalibaculum, Defluviitaleaceae_UCG-011), increased gastrin and motilin levels, decreased calcitonin gene-related peptide, upregulated mucin-2 mRNA, elevated fecal acetate and butyrate, improved small intestinal propulsion rate, and increased fecal output within 5 hours, demonstrating superior constipation relief. Conclusion: The study demonstrates that the combined prebiotics (containing 3 g xylooligosaccharides, 3 g stachyose, and 8 g galactooligosaccharides per 100 mL) effectively relieved constipation. Prebiotics combined with prune juice further enhancing efficacy through improved intestinal motility and microbiota modulation. These findings support the development of multi-component prebiotic formulations and reveal prune juice's potential to amplify prebiotic effects, providing a scientific basis for novel constipation management strategies.