Biotin, a water-soluble micronutrient, is vital for cellular functions, including growth and development. The human intestine utilizes the human sodium-dependent multivitamin transporter (hSMVT) for biotin uptake. Evidence exists showing that the intestinal biotin uptake process is adaptively regulated during biotin deficiency. Nothing, however, is known about molecular mechanism(s) involved during this adaptive regulation. This study compared two human-derived intestinal epithelial cell lines (HuTu-80 and Caco-2) during biotin-deficient or biotin-sufficient states and with an approach that assessed carrier-mediated biotin uptake, hSMVT protein and RNA levels, RNA stability, and hSMVT promoter activity. The results showed that during biotin deficiency, a significant and specific upregulation in carrier-mediated biotin uptake occurred in both human intestinal epithelial cell lines and that this increase was associated with an induction in protein and mRNA levels of hSMVT. The increase in mRNA levels was not due to an increase in RNA stability but was associated with an increase in activity of the hSMVT promoter in transfected human intestinal cells. Using promoter deletion constructs and mutational analysis in transiently transfected HuTu-80 and Caco-2 cells, a biotin deficiency-responsive region was mapped to a 103-bp area within the hSMVT promoter that contains gut-enriched Kruppel-like factor (GKLF) sites that confer the response to biotin deficiency. These results confirm that human intestinal biotin uptake is adaptively regulated and provide novel evidence demonstrating that the upregulation is not mediated via changes in hSMVT RNA stability but rather is due to transcriptional regulatory mechanism(s) that likely involve GKLF sites in the hSMVT promoter.