Cellular retinol-binding protein type II (CRBPII) is abundantly expressed in the small intestinal enterocytes of many vertebrates and plays important physiological roles in intestinal absorption, transport and metabolism of vitamin A. In the present study, we investigated regulation of human CRBPII gene expression using human intestinal Caco-2 BBe cells. We found that the human CRBPII gene contained a direct-repeat 1 (DR-1)-like nuclear receptor response element in the proximal promoter region, and that endogenous hepatocyte nuclear factor-4 (HNF-4) was a major transcription factor binding to the DR-1-like element. Co-transfection of HNF-4 expression vector transactivated the human CRBPII gene promoter activity, whereas mutation of the DR-1-like element abolished the promoter activity. Stably transfected Caco-2 BBe cells overexpressing HNF-4 significantly increased endogenous CRBPII gene expression and retinyl ester synthesis. Reduction of HNF-4 protein levels by HNF-4 siRNA decreased CRBPII gene expression. Caco-2 BBe cells treated with phorbol 12-myristate 13-acetate, a protein kinase C activator, decreased nuclear HNF-4 protein level and its binding activity to the human CRBPII gene DR-1-like element, as well as CRBPII gene expression. Moreover, nuclear HNF-4 protein levels, its binding to human CRBPII DR-1-like elements, and CRBPII gene expression level were coordinately increased during Caco-2 BBe cell differentiation. These results suggest that HNF-4 is an important transcriptional factor that regulates human CRBPII gene expression and provide possibility for a novel function of HNF-4 in the regulation of human intestinal vitamin A absorption and metabolism.