Intestinal alkaline phosphatase (IAP) is an enterocyte differentiation marker that functions to limit fat absorption. ZBP-89 is a Kruppel-type transcription factor that appears to promote a differentiated phenotype in the intestinal epithelium. The purpose of these studies was to investigate the regulation of IAP gene expression by the ZBP-89. Reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR, western analyses and reporter assays were used to determine the regulation of IAP by ZBP-89 in HT-29 and Caco-2 colon cancer cells. ZBP-89 knockdown was achieved by specific siRNA. Electrophoretic mobility shift assay (EMSA) and chromatin-immunoprecipitation (ChIP) were performed to examine binding of ZBP-89 to the IAP promoter. The results of RT-PCR, quantitative real-time PCR, and western blotting showed that ZBP-89 was expressed at low levels in Caco-2 and HT-29 cells, whereas IAP was minimally expressed and absent in these cells, respectively. Transfection with ZBP-89 expression plamid increased IAP mRNA and protein levels in both cell lines, whereas knockdown of the endogenous ZBP-89 by siRNA reduced the basal levels of IAP gene expression in Caco-2 cells. IAP-luciferase reporter assays, EMSA, and ChIP established that ZBP-89 activated the IAP gene through a response element (ZBPRE: 5'-CCTCCTCCC-3') located between -1018 and -1010 bp upstream of the AUG start codon. We conclude that ZBP-89 is a direct transcriptional activator of the enterocyte differentiation marker IAP. These findings are consistent with the role that this transcription factor is thought to play as a tumor suppressor, and suggests its possible function in the physiology of fat absorption.