Using both in vivo and in vitro models, we found that knocking down ASCT2 by shRNAs or miR-137 or the combination of silencing ASCT2 and pharmacologically inhibiting SNAT2 via a small-molecule antagonist called V-9302 significantly suppressed intracellular glutamine levels and downstream glutamine metabolism, including glutathione production; these effects attenuated growth and proliferation, increased apoptosis and autophagy, and increased oxidative stress and mTORC1 pathway suppression in HNSCC. This evidence concerns the gene SLC1A5 and head and neck squamous cell carcinoma.