Vitamin C, an essential micronutrient and a critical cofactor for several intracellular enzymatic reactions, is actively transported across epithelial barriers through the sodium-dependent ascorbic acid transporter 1 (SLC23A1). The SLC23A1 gene encodes SLC23A1 transporter and is located on human chromosome 5q31.2 (138702885-138719039 compl.; NC_000005.9). Previously, the SLC23A1 transcript has been described as containing 15 exons, stretching over 16 kilobases; however, no alternative splice variant has been described yet. Variations in SLC23A1 gene would impact vitamin C dietary requirements and/or susceptibility to common complex diseases. We have, therefore, evaluated the existence of additional SLC23A1 transcripts in silico and examined the function of new transcript through Xenopus laevis oocytes injection and the transporter expression. Here, we characterized a novel alternative first exon encoding a SLC23A1 isoform which is exclusive to humans and expressed in the small intestine. The novel SLC23A1 protein isoform adds 36 N-terminal amino acids but does not alter transmembrane topology or intracellular location. The novel SLC23A1 isoform is located on the plasma membrane and mediates very low ascorbic acid transport when expressed in Xenopus laevis oocytes. This is the first report of an alternative splice variant in human SLC23A1 locus and confirmed an extension of about 1kb further 5' than previously described gene. 