Monocarboxylate transporter 8 (MCT8) facilitates cellular uptake and efflux of thyroid hormone (TH). Mutations in MCT8 result in severe intellectual and motor disability known as the Allan-Herndon-Dudley syndrome (AHDS). Previous studies have provided valuable insights into the putative mechanism of substrate binding in the inward-open conformation, required for TH efflux. The current study aims to delineate the mechanism of substrate binding in the outward-open conformation, required for TH uptake. Extensive chemical modification and site-directed mutagenesis studies were used to guide protein homology modeling of MCT8 in the outward-open conformation. Arg271 and Arg445 were modified by phenylglyoxal, which was partially prevented in the presence of substrate. Substrate docking in our outward-open model suggested an important role for His192 and Arg445 in substrate binding. Interestingly, mutations affecting these residues have been identified in patients who have AHDS. In addition, our outward-open model predicted the location of Phe189, Met227, Phe279, Gly282, Phe287, and Phe501 at the substrate-binding center, and their Ala substitution differentially affected the apparent Vmax and Km of T3 transport, with F189A, F279A, and F287A showing the highest impact. Thus, here we present an MCT8 homology model in the outward-open conformation, which supports the important role of His192 and Arg445 in substrate docking and identifies critical residues at the putative substrate-binding center. Our findings provide insights into MCT8 structure and function, which add to our understanding of the pathogenic mechanism of mutations found in patients who have AHDS and can be used to screen for novel substrates and inhibitors.