To investigate effects of berberine exerts on A20 expression and regulation of intestinal epithelial tight junctions via the TNF-alpha-NF-kappa B-MLCK pathway in Diarrhea-Predominant Irritable Bowel Syndrome (IBS-D). C57BL/6 wild type (WT) and A20 IEC-KO mice (48 each) were randomly divided into normal control (NC), model control (MC), rifaximin and berberine groups (12 mice per group). An experimental model of IBS-D was established using 4% acetic acid and evaluated by haematoxylin-eosin (HE) staining. rifaximin and berberine mice were treated with rifaximin and berberine, respectively. Intestinal epithelial space of WT berberine mice improved more than A20 IEC-KO berberine mice compared to MC mice. WT berberine mice exhibited greater expression of A20 compared with MC mice(P < 0.01). TNF-alpha, NF-kappa B p65, MLCK, MLC, TRAF6 and RIP1 levels in A20 IEC-KO and WT berberine mice were all decreased compared to MC mice(P all < 0.05). NF-kappa B p65, MLCK and TRAF6 levels were increased in A20 IEC-KO berberine mice as compared to WT berberine mice (P all < 0.05). Intestinal epithelial levels of occludin, claudin-1, ZO-1 and F-actin increased in all berberine mice (P all < 0.01-0.05), while occludin, claudin-1, and ZO-1 levels were lower in A20 IEC-KO berberine mice(P < 0.05). Berberine downregulates abnormal activation of the TNF-alpha-NF-kappa B-MLCK pathway by upregulating expression of A20 in a mouse model of IBS-D, thereby protecting intestinal epithelial tight junctions and repairing the damage IBS-D causes to the intestinal epithelial barrier.