Vitamin C is transported as ascorbic acid (AA) through the sodium-ascorbate cotransporters (SVCT1 and -2) and as dehydroascorbic acid (DHA) through the facilitative glucose transporters. All cells have glucose transporters and take up DHA that is trapped intracellularly by reduction and accumulated as AA. SVCT2 is widely expressed in cells and tissues at the mRNA level; however, only specialized cells directly transport AA. We undertook a molecular analysis of SVCT2 expression and discovered a transcript encoding a short form of human SVCT2 (hSVCT2-short) in which 345 bp is deleted without a frame shift. The deletion involves domains 5 and 6 and part of domain 4. cDNA encoding this isoform was isolated and expressed in 293T cells, where the protein was detected on the plasma membrane. Transport studies, however, revealed that hSVCT2-short gave rise to a nonfunctional transporter protein. hSVCT2-short arises by alternative splicing and encodes a protein that strongly inhibited the function of SVCT2 and, to a lesser extent, SVCT1 in a dominant-negative manner, probably by protein-protein interaction. The expression of hSVCT2-short varies among cells. PCR analysis of cDNA isolated from melanocytes capable of transporting AA revealed a predominance of the full-length isoform, while HL-60 cells, which express SVCT2 at the mRNA level and were incapable of transporting AA, showed a predominance of the short isoform. These findings suggest a mechanism of AA uptake regulation whereby an alternative SVCT2 gene product inhibits transport through the two known AA transporters.