Cellular and molecular regulation of the renal biotin uptake in human is not well defined. In addition, the contribution of the human sodium-dependent multivitamin transporter, hSMVT, toward carrier-mediated biotin uptake by human proximal tubular epithelial cells is not clear. The aims of this study were, therefore, to address these issues using the human-derived proximal tubular epithelial HK-2 cells as a model. First, we characterized the mechanism of biotin uptake by these cells and obtained evidence for the involvement of a Na⁺, temperature- and energy-dependent carrier-mediated uptake system. This system was inhibited by the biotin structural analogue desthiobiotin, pantothenic acid and lipoate. These findings suggest the involvement of the hSMVT system in the uptake process. This was confirmed by demonstrating that the hSMVT system is indeed expressed in HK-2 cells at the protein and mRNA levels, and by selectively silencing the hSMVT gene with the use of gene-specific small interfering RNAs (siRNAs) which lead to specific and significant inhibition in carrier-mediated biotin uptake. Of the two recently cloned promoters of the hSMVT gene, promoter 1 was found to be more active than promoter 2 in these cells. Pre-treatment of the HK-2 cells with modulators of protein kinase C (PKC) and Ca²⁺/calmodulin (Ca²⁺/CaM)- mediated pathways [but not those that modulate protein kinase A (PKA), protein tyrosine kinase (PTK), or nitric oxide(NO)- mediated pathways] lead to significant alterations in biotin uptake. Maintaining the HK-2 cells in a biotin-deficient growth medium lead to a marked up-regulation in biotin transport, which was associated with an increase in hSMVT protein and RNA levels as well as by an increase in the activity of the hSMVT promoters. These results demonstrate that biotin uptake by human renal epithelial cells occurs via the hSMVT system and that the process is under the regulation of an intracellular PKC and Ca²⁺/CaM-mediated pathways. In addition, the uptake process appears to be adaptively regulated by extracellular biotin level and that this regulation involves transcriptional regulatory mechanism(s).