The mechanism of biotin uptake in human intestine has been well characterized and involves the human sodium dependant multivitamin transporter (hSMVT), yet little is known about the molecular/transcriptional regulation of the system. Previous investigations cloned the 5' regulatory region of the hSMVT gene and identified the minimal promoter. To expand these investigations we compared activity of the hSMVT promoter in three human intestinal epithelial cell lines (NCM460, Caco-2, and HuTu-80) and contrasted a renal epithelial cell line (HEK293). We analyzed the role of putative cis-elements in regulating promoter activity and confirmed activity of the cloned hSMVT promoter in vivo. In vitro studies demonstrated that all cell lines utilized the same minimal promoter region and mutation of specific cis-regulatory elements (KLF-4 and AP-2) led to a decrease in promoter activity in all intestinal cell types but not in renal cells. Using electrophoretic mobility shift assays (EMSA) two specific DNA/protein complexes were identified. Oligonucleotide competition and antibody supershift analysis determined that KLF-4 and AP-2 were involved in forming the complexes. In HEK293 cells over-expressing KLF-4 increased the endogenous hSMVT message levels three fold and activated a co-transfected hSMVT promoter-reporter construct. In vivo studies using hSMVT promoter-luciferase transgenic mice established physiological relevance and showed the pattern of hSMVT promoter expression to be similar to endogenous mouse SMVT mRNA expression. The results demonstrate for the first time, the importance of KLF-4 and AP-2 in regulating the activity of the hSMVT promoter in the intestine and provide direct in vivo confirmation of hSMVT promoter activity.