Diarrhea-predominant irritable bowel syndrome (IBS-D) is a common chronic functional gastrointestinal disorder. MicroRNAs (miRNAs) have been identified to be involved in different physiological and pathological processes. In this study, the role of miRNA-29a in the potential mechanism underlying the function of the intestinal mucosal barrier in IBS-D was analyzed. Human intestinal mucosal epithelia from patients with IBS-D (diagnosed as meeting the Rome IV criteria) and healthy volunteers were collected. An IBS-D mouse model was established via induction with trinitro-benzene-sulfonic acid (TNBS), and the mice were injected with miRNA-29a inhibitor. Using transmission electron microscopy (TEM), the epithelial ultrastructure of the human intestinal mucosa was examined. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, the expression level of miRNA-29a was assessed. ELISA was used to analyze the activity of D-lactate (D-LA) and diamine oxidase (DAO). Through immunohistochemistry, RT-qPCR and western blotting, the expression of tight junction protein ZO-1 (ZO-1) and claudin-1 (CLDN1) was examined. In the human intestinal mucosal epithelia from patients with IBS-D, miRNA-29a was upregulated, ZO-1 and CLDN1 were downregulated, and the junctional complex (JC) was faint and discontinuous. In the IBS-D mouse model, treatment with miRNA-29a inhibitor downregulated D-LA and DAO activity, and increased the expression of ZO-1 and CLDN1 in the intestinal mucosal epithelium. In conclusion, the present study revealed that miRNA-29a is involved in the pathogenesis of IBS-D, probably by downregulating ZO-1 and CLDN1 expression, suggesting that miRNA-29a is likely to be an important regulator of intestinal barrier function and could be a possible therapeutic target for IBS-D. Copyright: © Zhu et al.