Background/Objectives: Irritable bowel syndrome (IBS) is a highly prevalent functional gastrointestinal disorder. Emerging evidence implicates gut microbiota dysbiosis in IBS pathogenesis, and probiotic interventions targeting microbial modulation hold therapeutic promise. Methods: this study used fecal microbiota transplantation to establish a mouse model of IBS before evaluating the effects of the complex probiotic by using metagenomics and targeted metabolomics to explore the potential mechanism. Results: After 14 days, the probiotic relieved constipation, reduced inflammation and intestinal permeability, lowered 5-HT levels and increased serotonin transporter (SERT) expression in tissues. Metagenomic analysis showed a reduced inflammation-related species abundance. It also decreased fecal butyric acid, acetic acid and tryptophan levels in IBS mice. Conclusions: The probiotic complex effectively alleviated IBS symptoms in mice by modulating gut microbiota and fecal metabolites, providing insights for future IBS research and treatment.