Irritable bowel syndrome (IBS) is a common clinical functional gastrointestinal disease. It has a complex pathophysiological mechanism, in which the imbalance of gut microbiota might play an important role. Lacidophilin tablets (LH) can regulate gut microbiota, but their effect on IBS is unknown. In this study, the IBS model was established by acetic acid enema combined with the constrained stress method, and rats were fed LH for 2 weeks. LH significantly reduced visceral sensitivity and intestinal propulsion rate and improved IBS-induced anxiety and depressive behavior in IBS rats. LH elevated the expression levels of mucin 2, claudin1, and occludin, and ameliorated IBS-induced structural damage to colonic tissues. The gut microbiota analysis revealed that LH altered the structure and composition of the gut microbiota in IBS rats. In addition, LH reduced the expression levels of inflammatory factor-related genes. These results suggest that LH could significantly improve the visceral sensitivity and intestinal motility disorders of IBS rats, relieve anxiety and depression levels, and alleviate the symptoms of IBS rats by regulating gut microbiota and reducing intestinal inflammation.