The molecular mechanisms that regulate the proliferation and differentiation of intestinal mucosal epithelial cells are not well understood. Keratinocyte growth factor (KGF) is an epithelial cell-specific growth factor that may be involved in the maintenance of mucosal epithelial populations and in mediating epithelial repair after injury. The sucrase-isomaltase (SI) gene, which encodes an enterocyte brush border disaccharidase, has served as a model for study of intestinal-specific gene expression and differentiation. KGF down-regulated SI mRNA and protein expression in Caco-2 intestinal epithelial cells but not the expression of other brush border enzymes. The down-regulation was dose- and time-dependent and specifically blocked by anti-KGF antibodies. Transfection experiments using SI promoter constructs demonstrated that KGF decreased SI gene transcription. In contrast, the stability of SI mRNA was not affected by incubation of Caco-2 cells with KGF. Electrophoretic mobility shift analysis demonstrated that binding of nuclear proteins to the SI footprint (SIF) 3 and SIF4 regulatory elements within the SI promoter region was increased in Caco-2 cells that had been incubated with KGF. In transfection experiments using a construct in which tandem copies of the SIF4-binding site were inserted upstream of the SV40 promoter and luciferase gene, incubation with KGF resulted in a significant decrease in luciferase activity. However, transfection with a similar construct containing tandem copies of SIF3 had no significant effect on SV40 promoter activity following KGF treatment. SIF4 may bind E4BP4, a previously identified transcriptional repressor protein. This factor may in part mediate the decrease in SI transcription by KGF in Caco-2 cells.