Studies on colorectal carcinogenesis have suggested a critical role of the adenomatous polyposis coli (APC) gene in the development of colorectal cancer. Gut-enriched Krüppel-like factor (GKLF) is a zinc-finger transcription protein with high expression in the colonic epithelium. Our previous studies have shown that GKLF transcript was significantly decreased in colon cancer tissue and suggested that it might play a role in the tumorigenesis of the colon. The signaling pathway of GKLF-regulated cell growth is currently unknown. We sought to determine if the functions of GKLF are mediated through the APC/beta-catenin pathway. In a colon cancer cell line (HT29-APC), containing a zinc-inducible APC gene, GKLF mRNA levels were significantly increased when wild-type APC protein was induced. No effect on GKLF mRNA concentration was observed in a control cell line (HT29-beta-gal), containing an analogous inducible lacZ gene. GKLF promoter activity was induced by co-transfection with wild-type APC DNA, suggesting that APC might be involved in transcriptional activation of the GKLF gene. In HT-29 cells, overexpression of GKLF resulted in decreases in beta-catenin protein and mRNA levels and down-regulation of GKLF expression led to increase in beta-catenin concentration. Overexpression of GKLF in HT-29 cells inhibited DNA synthesis and this effect was attenuated by co-transfection with wild-type beta-catenin, suggesting an essential role of beta-catenin in GKLF signaling. Furthermore, co-transfection of GKLF in colon cancer SW 480 cells abrogated the transcriptional activity of a beta-catenin-T-cell factor (Tcf) reporter construct in a dose-dependent manner. These findings indicate that the growth-suppressive effect of GKLF may be mediated through the APC/beta-catenin pathway. We speculate that when APC is mutated, GKLF gene expression is down-regulated, resulting in increases in beta-catenin level and transactivation of growth-promoted genes.