Endotoxemia is the crucial factor contributes to Alcoholic liver disease (ALD), and the disruption of intestinal tight junction caused by alcohol is one of the main underlying mechanisms. Berberine is used wildly to treat gastrointestinal disorders and was reported that it could restore the gut permeability. This study aimed to investigate whether berberine could improve intestinal epithelial permeability increased by alcohol and the pathway. Caco-2 cells monolayer were treated with various concentration of alcohol to induce barrier damage, and the effect of berberine on tight junction was analyzed by measuring the transepithelial electrical resistance (TEER). Subcellular localization of tight junction protein ZO-1 was observed by immuno?uorescence microscopy. The expression level of ZO-1, myosin light chain kinase (MLCK) and MLCK activation were analyzed by Western blot. And intracellular calcium influx was measured by flow cytometry. The results showed that berberine treatment could significantly down-regulate epithelial permeability increased by alcohol, up-regulate ZO-1 protein expression, improve intracellular calcium influx, and inhibit phosphorylation of MLCK in intestinal epithelium. According to results, we concluded that berberine can ameliorate alcohol induced intestinal epithelial tight junction damage in vitro, and this function may be through inhibiting MLCK signaling pathway. So berberine may be one effective therapeutic agent to attenuate the endotoxemia in ALD.