Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder marked by abdominal pain and irregular bowel habits. Recently, more and more evidence supports gut microbiota imbalance in IBS and highlights the potential of probiotics in restoring gut health and reducing symptoms. In this study, we explored the effects of PS133 (PS133) on an IBS-like condition in rats triggered by 5-hydroxytryptophan (5-HTP), a serotonin precursor. Eight-week-old Sprague Dawley rats received either PS133 or saline for 14 days, followed by 5-HTP to induce IBS-like symptoms. Colorectal distension tests showed that PS133 reduced visceral hypersensitivity. PS133 also protected intestinal mucin against 5-HTP-induced degradation, as seen in alcian blue staining, and increased the levels of tight junction proteins (occludin and zonula occludens-1) in the colon, indicating improved gut barrier integrity. Additionally, PS133 normalized the levels of substance P (a neuropeptide) in the spinal cord and altered 5-hydroxyindoleacetic acid (a serotonin metabolite) in the brain. Gut microbiota analysis revealed PS133 regulated specific bacterial groups, including [] and . Overall, PS133 improved gut function, reduced IBS-like symptoms, and modulated gut microbiota, neurotransmitters, and intestinal barrier health in this IBS model.