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Nutrient transporters expressed on cell membrane have been targeted for enhancing bioavailability of poorly permeable drugs. Sodium dependent multivitamin transporter (SMVT) is once such carrier system, utilized for improving drug targeting to specific tissues. Therefore, the main objective of this study is to characterize SMVT in human derived prostate cancer cells (PC-3). Reverse transcription polymerase chain reaction (RT-PCR) analysis has provided product band at 774 bp, specific to SMVT. The mechanism and intracellular regulation of [3H]-biotin is also studied. [3H]-biotin uptake is found to be time and concentration dependent with Km and Vmax values of 19 ± 2 μM and 23 ± 1 pmol/min/mg protein, respectively. The uptake process is saturable in micromolar concentration range but linear in nanomolar concentration range. [3H]-biotin uptake shows significant sodium, temperature, pH and energy dependency. The process is strongly inhibited by unlabeled biotin and structural analogs such as desthiobiotin, pantothenate, lipoate and valeric acid. Intracellular regulatory pathways such as Ca2+/calmodulin and PKC pathway but not PTK pathway appears to play an important role in modulating [3H]-biotin uptake. This study for the first time confirms the molecular expression of SMVT and demonstrates that SMVT, responsible for biotin uptake is functionally active in PC-3 cells.