Gut-enriched Krüppel-like factor (GKLF or KLF4) is a zinc finger-containing, epithelial-specific transcription factor, that functions as a suppressor of cell proliferation. We previously showed that GKLF expression is decreased in intestinal and colonic adenomas, respectively, from multiple intestinal neoplasia (Min) mice and familial adenomatous polyposis (FAP) patients. This study shows that GKLF is induced upon activation of the adenomatous polyposis coli (APC) gene. However, among several human colon cancer cell lines surveyed, expression of GKLF is lowest in RKO, a line with wild-type APC and β-catenin. RKO contains a mutated allele that encodes the putative tumor suppressor homeodomain protein, CDX2. We show that wild-type CDX2 activates the GKLF promoter and that the mutated CDX2 has a dominant negative effect on wild-type function. Our results may help explain the exceedingly low levels of GKLF expression detected in this cell line, which may in turn contribute to the tumor phenotype.