Constipation is a common intestinal disorder linked to an imbalance in the gut microbiota, which can be alleviated by synbiotics as a blend of probiotics and prebiotics. In this study, three strains of Lactiplantibacillus plantarum AHQ-14, HM22 and Y12, which have shown to have constipation-relieving ability, and four prebiotics including stachyose, galactooligosaccharides, isomaltooligosaccharide and fructooligosaccharides, were selected to design a synbiotic named AHY. Its intervention in vivo experiment using loperamide-induced constipated mice was performed. Relevant biochemical indexes, histological morphology, metabolites, constipation-related gene expression and intestinal flora of loperamide-induced constipated mice were investigated to elucidate the potential mechanism for AHY relieving constipation. The results showed that AHY increased the abundance of the flora associated to short-chain fatty acids (SCFAs) and water pathway protein, such as Muribaculaceae and Prevotellaceae, thereby the content of propionic acid and isovaleric acid were increased by 56.8% and 20%, respectively, and the expression of water pathway protein genes was significantly down-regulated. It was also found that AHY significantly increased the serum levels of excitatory gastrointestinal regulatory peptides GAS and MTL, and decreased the levels of inhibitory gastrointestinal regulatory peptide VIP and oxidative stress indicators MDA and NO in constipated mice. Furthermore, it significantly increased the fecal water content and gastrointestinal motility, and restored the colonic damage, thus alleviating the symptoms of constipation. These findings suggest AHY has the potential used as a promising functional synbiotic for constipation relief.