It has been reported that the traditional Chinese medicine Huangqin-Tang decoction (HQT) has a protective effect on the epithelial barrier function of ulcerative colitis, but its mechanism has not been fully clarified. This study intends to explore the protective mechanism of HQT in regulating microRNA (miRNA) for the first time.Based on the Balb/c mice ulcerative colitis model, the mice were given a gavage of 0.1 mL/10 g HQT every day for 7 days; on the 8th day, the colon of the mice was dissected, the length of the colon for the mice was measured, and the score was given based on this. Analysis of colonic mucosal injury was conducted by hematoxylin-eosin staining. Then, the differential miRNA was screened and sequenced in colon tissue using the HiSeq platform. And the differential miR-185-3p gene was verified by RT-PCR. Finally, the effects of HQT on miR-185-3p, occludin protein expression, and transepithelial electrical resistance (TEER) value were observed in combination with the CaCo2 intestinal epithelial cell model.HQT treatment can alleviate the shortening of colon length and reverse the intestinal mucosal injury. miRNA sequencing of colonic tissue showed that miR-185-3p was significantly downregulated in the model group, while HQT could upregulate miR-185-3p, thereby affecting the myosin light chain kinase (MLCK)/myosin light chain phosphorylation (p-MLC) pathway and leading to increased expression of occludin protein, which ultimately protected the intestinal epithelial barrier function.HQT can protect colon epithelial barrier function by regulating miR-185-3p.Copyright © 2021 Zhou Changlin et al.