The study evaluated the postbiotic Probio-Eco for chronic constipation using a randomized, double-blind, placebo-controlled crossover trial in adults and a parallel mouse model. In humans, a 3-week Probio-Eco intervention significantly increased complete spontaneous bowel movements, improved stool consistency, and reduced straining compared to placebo. Multi-omics profiling showed that the postbiotic altered gut microbiota composition and increased key metabolites such as succinate, indole derivatives, and 5-HT-related compounds, which correlated with symptom relief. Mouse experiments confirmed that these metabolites enhanced colonic mucus secretion, improved gut-motility hormones, reduced inflammation, and restored intestinal transit. Overall, the postbiotic improved constipation by modulating the microbiota–metabolite–gut-motility axis.