The aim of this study was to investigate expression and relative contribution of human thiamin transporter (hTHTR)-2 toward overall carrier-mediated thiamin uptake by human intestinal epithelial cells. Northern blot analysis showed that the message of the hTHTR-2 is expressed along the native human gastrointestinal tract with highest expression being in the proximal part of small intestine. hTHTR-2 protein was found, by Western blot analysis, to be expressed at the brush-border membrane (BBM), but not at the basolateral membrane, of native human enterocytes. This pattern of expression was confirmed in studies using a fusion protein of hTHTR-2 with the enhanced green fluorescent protein (hTHTR2-EGFP) expressed in living Caco-2 cells grown on filter. Pretreating Caco-2 cells (which also express the hTHTR-2 at RNA and protein levels) with hTHTR-2 gene-specific small interfering RNA (siRNA) led to a significant (P < 0.01) and specific inhibition (48%) in carrier-mediated thiamin uptake. Similarly, pretreating Caco-2 cells with siRNA that specifically target hTHTR-1 (which is expressed in Caco-2 cells) also significantly (P < 0.01) and specifically inhibited (by 56%) carrier-mediated thiamin uptake. When Caco-2 cells were pretreated with siRNAs against both hTHTR-2 and hTHTR-1 genes, an almost complete inhibition in carrier-mediated thiamin uptake was observed. These results show that the message of hTHTR-2 is expressed along the human gastrointestinal tract and that expression of its protein in intestinal epithelia is mainly localized to the apical BBM domain. In addition, results show that this transporter plays a significant role in carrier-mediated thiamin uptake in human intestine.