In KLF4 deficient mice, colonic goblet cell numbers are significantly reduced. Goblet cell development is regulated by the Notch signaling pathway. The aim of this study was to examine whether Notch represses KLF4 expression to regulate goblet cell differentiation. We first detected KLF4 gene expression was upregulated in a human progastrin-overexpressing mouse model where goblet cell hyperplasia has been observed. We then found that mice treated with a gamma-secretase inhibitor (Compound E, 10µmol/kg) for 24 hours which inhibits the Notch signaling pathway had significantly increased KLF4 mRNA levels in small intestine and colon, accompanied by increased number of KLF4 expressing cells at the bottom of crypts in small intestine and colon. In a colon cancer cell line (HCT116 cells), KLF4 promoter activity was inhibited by a constitutively active form of Notch1 (ICN1) by transient cotransfection assays. This inhibition was significantly compromised by a dominant negative RBPjk, a repressive mediator of the Notch signaling pathway. An ICN1 responsive element was then mapped in the human KLF4 promoter between -151 and -122 nucleotides upstream of the transcriptional start site. It was also found that an intact ICN1 responsive element is required for ICN1 to inhibit KLF4 promoter activity by transient cotransfection assays. Our findings thus reveal a possible mechanism by which KLF4 is inhibited by Notch, which controls goblet cell differentiation in mouse gastrointestinal tract.