The glutamine transporter ASCT2 is highly overexpressed in cancer cells. Block of glutamine uptake by ASCT2 is a potential strategy to inhibit growth of cancer cells. However, pharmacology of the ASCT2 binding site is not well established. In this work, we report the computational docking to the binding site, and the synthesis of a new class of ASCT2 inhibitors based on the novel L-hydroxyhomoserine scaffold. While these compounds inhibit the ASCT2 leak anion conductance, as expected for competitive inhibitors, they did not block leak conductance in glutamate transporters (EAAT1-3 and EAAT5). They were also ineffective with respect to subtype ASCT1, which has >57% amino acid sequence similarity to ASCT2. Molecular docking studies agree very well with the experimental results and suggest specific polar interactions in the ASCT2 binding site. Our findings add to the repertoire of ASCT2 inhibitors and will aid in further studies of ASCT2 pharmacology.