Nucleoside transporters are plasma membrane proteins essential for nucleoside salvage. Among them, human concentrative nucleoside transporter 3 (hCNT3, SLC28A3) plays an essential role in this process because of its broader substrate selectivity and higher concentrative ability than the other members of the SLC28 protein family, hCNT1 and hCNT2. The aim of this study was to characterize an isoform of hCNT3, encoded by an alternatively spliced SLC28A3-related mRNA, the first identified for a CNT protein. This variant, named hCNT3ins, is the result of the insertion of 176 bp corresponding to an intron located between exons 2 and 3 of the gene. This insertion results in a shift of the reading frame, yielding a protein lacking 69 residues of the N terminus. hCNT3 and hCNT3ins mRNAs are simultaneously expressed both in normal and transformed cells and are differentially regulated by activation and differentiation. Because of the N-terminal deletion, hCNT3ins is retained in the endoplasmic reticulum, where it shows a typical hCNT3-related activity. hCNT3ins exhibits a shorter half-life than its plasma membrane counterpart, being degraded via a proteasome-dependent pathway. We suggest that this novel hCNT3 isoform would be involved in the salvage of intracellular nucleosides from the lumen of the endoplasmic reticulum to the cytoplasm.—Errasti-Murugarren, E., Molina-Arcas, M., Casado, F. J., Pastor-Anglada, M. A splice variant of the SLC28A3 gene encodes a novel human concentrative nucleoside transporter-3 (hCNT3) protein localized in the endoplasmic reticulum.