Sucrase-isomaltase (SI), an intestine-specific gene, is induced in the differentiated small intestinal villous epithelium during the suckling-weaning transition in mice. We have previously identified cis-acting elements within a short evolutionarily conserved SI promoter. However, the nature and profile of expression of the interacting proteins have not been fully characterized during this developmental transition. Herein, we show that hepatocyte nuclear factor-1 alpha (HNF-1 alpha), GATA-4, and caudal related homeodomain proteins Cdx2 and Cdx1 are the primary transcription factors from the adult mouse intestinal epithelium to interact with the SIF3, GATA, and SIF1 elements of the SI promoter. We wanted to study whether HNF-1 alpha, GATA-4, and Cdx2 can cooperate in the regulation of SI gene expression. Immunolocalization experiments revealed that HNF-1 alpha is detected in rare epithelial cells of suckling mice and becomes progressively more expressed in the villous epithelial cells during the suckling-weaning transition. GATA-4 protein is expressed exclusively in villous differentiated epithelial cells of the proximal small intestine, decreases in expression in the ileum, and becomes undetectable in the colon. HNF-1 alpha, GATA-4, and Cdx2 interact in vitro and in vivo. These factors activate SI promoter activity in cotransfection experiments where GATA-4 requires the presence of both HNF-1 alpha and Cdx2. These findings imply a combinatory role of HNF-1 alpha, Cdx2, and GATA-4 for the time- and position-dependent regulation of SI transcription during development.