Gut-enriched Krüppel-like factor (GKLF, KLF4) is an epithelial-specific transcription factor that expresses in the gastrointestinal tract and mediates growth arrest of colonic epithelium. The molecular mechanisms governing its growth inhibitory effect have not been fully elucidated. In the present study, we showed that induction of GKLF mRNA and protein expression by interferon-gamma treatment was associated with reduction of ornithine decarboxylase (ODC) gene expression and enzyme activity in colon cancer HT-29 cells. Overexpression of GKLF in HT-29 cells significantly reduced ODC mRNA and protein levels as well as enzyme activity and resulted in growth arrest, indicating that ODC might be a downstream target of GKLF. This conclusion was further supported by data showing that GKLF mRNA and protein concentrations were the highest at the G(1)/S boundary of the cell cycle, where ODC mRNA and protein levels were the lowest and that overexpression of GKLF resulted in cell arrested at the G(1) phase. Reporter gene transfection studies and electrophoretic mobility gel shift assays demonstrated that GKLF repressed ODC promoter activity and that these effects appeared to be mediated through interaction with a GC box in the proximal portion of the promoter. Transfection studies using reporter constructs and chromatin immunoprecipitation assays also demonstrated that GKLF inhibited transactivation of the ODC gene by interfering with the binding of Sp1 to the ODC promoter. These results indicate that GKLF may function as a G(1)/S checkpoint regulator and exert its growth arrest effect through down-regulation of ODC gene expression. Furthermore, GKLF is a transcriptional repressor of the ODC gene, and these effects are mediated by interaction with the GC-rich region on the promoter.