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Recent cryoEM studies elucidated details of the structural basis for the substrate selectivity and translocation of heteromeric amino acid transporters. However, Asc1/CD98hc is the only neutral heteromeric amino acid transporter that can function through facilitated diffusion, and the only one that efficiently transports glycine and D-serine, and thus has a regulatory role in the central nervous system. Here we use cryoEM, ligand-binding simulations, mutagenesis, transport assays, and molecular dynamics to define human Asc1/CD98hc determinants for substrate specificity and gain insights into the mechanisms that govern substrate translocation by exchange and facilitated diffusion. The cryoEM structure of Asc1/CD98hc is determined at 3.4–3.8 Å resolution, revealing an inward-facing semi-occluded conformation. We find that Ser 246 and Tyr 333 are essential for Asc1/CD98hc substrate selectivity and for the exchange and facilitated diffusion modes of transport. Taken together, these results reveal the structural bases for ligand binding and transport features specific to human Asc1., The CNIO Electronic Microscopy Unit is acknowledged for support in cryoEM sample screening and initial data acquisition. CryoEM data used in this work was obtained at the Cryo-Electron Microscopy Facility at University of Leicester and at the Diamond Light Source cryo-EM facility at the UK’s National Electron Bio-imaging Center (eBIC) under BAG Proposal No BI26876 “PID20410 VID35329 - Stop cancer - structural studies of macromolecular complexes involved in cancer by cryo-EM”. We also acknowledge the IRB-Barcelona core facilities of Protein Expression for support in cloning and expression of Asc1/CD98hc heterodimer, Advanced Digital Microscopy for support in immunofluorescence studies and Biostatistics/Bioinformatics for the statistical analysis of the data. This research was funded by La Caixa (LCF/PR/HR20/52400017 to O.L. and M.P.) and by the Spanish Ministry of Science and Innovation grant (PID2021-122802OB-I00 to M.P). Work in the Llorca lab is additionally funded by the Agencia Estatal de Investigación (AEI/10.13039/501100011 033 to O.L.); Ministerio de Ciencia e Innovación, (PID2020-114429RB-I00 to O.L.); O.L. laboratory also had the support from the National Institute of Health Carlos III to CNIO. Grant Carmen De Torres-Institut de recerca Sant Joan de Déu supports R.A. research. Paola Bartoccioni is supported by CIBERER-ISCIII., Peer Reviewed, "Article signat per 13 autors/es: Josep Rullo-Tubau, Maria Martinez-Molledo, Paola Bartoccioni, Ignasi Puch-Giner, Ángela Arias, Suwipa Saen-Oon, Camille Stephan-Otto Attolini, Rafael Artuch, Lucía Díaz, Víctor Guallar, Ekaitz Errasti-Murugarren, Manuel Palacín & Oscar Llorca", Postprint (published version)