Lactase-phlorizin hydrolase gene expression is spatially restricted along the anterior-posterior gut axis. Lactase gene transcription is maximal in the distal duodenum and jejunum in adult mammals and is barely detectable in the proximal duodenum. By contrast, pancreatic duodenal homeobox 1 protein, PDX-1, is expressed maximally in the proximal duodenum. The study aimed to determine the role of PDX-1 in regulating lactase gene promoter activity in intestinal epithelial cells. Caco-2 cells were co-transfected with lactase promoter-reporter constructs in the presence of a PDX-1 expression vector and assayed for luciferase activity. PDX-1 cotransfection results in repression of lactase promoter activity. Sequence analysis of the lactase promoter revealed a putative PDX-1 DNA binding site in the proximal 100 bp lactase gene promoter. Mobility shift assays demonstrated that PDX-1 can interact with the lactase promoter binding site but not with a site in which the core PDX-1 binding sequence, TAAT, is mutated. Site-directed mutagenesis of the PDX-1 core binding site in the lactase promoter-reporter construct suggests that PDX-1 can function independent of DNA binding to its consensus binding site. Stable over-expression of PDX-1 results in repression of the endogenous human lactase gene in differentiated Caco-2 cells. Given the contrasting spatial expression pattern, PDX-1 may function to specify the anterior boundary of lactase expression in the small intestine and is thus a candidate regulator of anterior spatial restriction in the gut.