High-circulating IGF-I concentrations are associated with an increased risk for breast, prostate, and colorectal cancer. Krüppel-like factor-6 (KLF6) is a zinc finger tumor suppressor inactivated in prostate and other types of cancer. We have previously demonstrated that KLF6 is a potent transactivator of the IGF-I receptor promoter. The aim of the present study was to examine the potential regulation of KLF6 gene expression by IGF-I. The human colon cancer cell lines HCT116 +/+ and -/- (with normal and disrupted p53, respectively) were treated with IGF-I. Western blots, quantitative RT-PCR, and transfection assays were used to evaluate the effect of IGF-I on KLF-6 production. Signaling pathway inhibitors were used to identify the mechanisms responsible for regulation of KLF6 expression. Small interfering RNA against p53 and KLF6 was used to assess the role of p53 in regulation of KLF6 expression by IGF-I and to evaluate KLF6 involvement in cell cycle control. Results obtained showed that IGF-I stimulated KLF-6 transcription in cells with normal, but not disrupted, p53, suggesting that KLF6 is a downstream target for IGF-I action. Stimulation of KLF6 expression by IGF-I in a p53-dependent manner may constitute a novel mechanism of action of IGF-I, with implications in normal cell cycle progression and cancer biology.