A33 antigen is a membrane-bound protein that is expressed only in intestinal epithelium and in most human colon cancers. Thus, A33 antigen has been explored as a potential therapeutic target for the treatment of colon cancers. However, little is known about the mechanism responsible for the tissue-specific pattern of its expression. In this report, we demonstrate that gut-enriched Krüppel-like factor (GKLF) binds to the promoter region of A33 antigen gene in colonic carcinoma cells and that mutations in the GKLF binding sequence in this region lead to diminished expression of A33 antigen. In addition, the expression of GKLF is linked to the expression of A33 antigen and blocking the expression of GKLF leads to the abolishment of A33 antigen expression. These results suggest that GKLF is a critical regulator in inducing the expression of A33 antigen in intestinal epithelium. While it has been suggested that GKLF is a regulator in inducing cell growth arrest and differentiation of the intestine, our observation that A33 antigen gene is a downstream target for GKLF suggests a more complex and diverse role for GKLF in the gut.