SLC19A2 is a membrane thiamine transporter that is expressed in variety of human tissues including the gastrointestinal tract. Little is currently known about the structure/function relationship of SLC19A2. In this study, we examined the effect of introducing mutations in SLC19A2 that are identical to those found in patients with thiamine-responsive megaloblastic anemia syndrome (TRMA), on functional activity and membrane expression of the transporter. We also examined the effect of mutating the only conserved anionic residue (E138) in any of the transmembrane domains of the SLC19A2 and that of the putative glycosylation sites (N63 and N314). Northern blot analysis showed thatSLC19A2 mRNA was expressed at the same level in HeLa cells transfected with wild-type or mutatedSLC19A2. Introducing the clinically relevant mutations (D93H, S143F, G172D) or mutation at the conserved anionic residue (E138A) of SLC19A2 led to a significant (p < 0.01) inhibition of thiamine uptake. Mutations of the two potential N-linked glycosylation sites (N63Q, N314Q) of SLC19A2 did not affect functional activity; they did however lead to a noticeable reduction in the apparent molecular weight (MW) of the protein. Western blot analysis showed that all proteins (except D93H) were expressed in the membrane (not the cytoplasmic) fraction of HeLa cells. These results provide direct confirmation that the clinically relevant mutations in SLC19A2 observed in TRMA patients causes mal-functioning of the transporter and/or a defect in its translation/stability. The results also show that the conserved transmembrane anionic residue of the SLC19A2 protein is critical for its function. Furthermore, native SLC19A2 appears to be glycosylated, but this appears to be not important for its function.