In summary, by knocking down endogenous KLK1 with AAV and combining it with the 2% DSS‐induced acute colitis model, we found that as inflammation intensified, the expression of KLK1 in colon tissue, serum, and intestinal epithelium further decreased, the intestine lost the protective effect of KLK1, and intestinal barrier markers further decreased. This evidence concerns the gene KLK1 and colitis.