Multiple gastrointestinal disorders are associated with impaired gut microbiota. Probiotic can improve bowel disorder, however, the action mechanism is poorly understood. We integrated multi-omics data from the gut metagenome, metabolome, and colon transcriptome of constipated mice underlying LRJ-1 treatment to provide insights into host-microbial metabolic pathway. We found that oral administration of LRJ-1 alleviated constipation in mice accompanied by the increased abundances of fecal γ-aminobutyric acid (GABA) and intestinal commensal , and the activation of host GABAergic synapses. was the most enriched species in constipated mice treated with LRJ-1, and contributed to the increased abundance of GABA in the gut. Administration of either ATCC 8492 or GABA alleviated constipation and increased gastrointestinal motility in constipated mice. Knockout of the GABA biosynthetic gene in ATCC 8492 decreased GABA production and blocked its beneficial effects on constipation. These results confirm the therapeutic potential of LRJ-1 in alleviating constipation through promoting gut commensal -derived GABA production. Targeting the gut microbiome to regulate GABA production may open new insights for efficient constipation treatment.