In this study, we observed that EBV upregulates the expression of two GLS1 isoforms, KGA and GAC, in EBV-positive lymphoma and nasopharyngeal cancer cells, and their localization to the mitochondria increases glutaminolysis and its end product, alpha-ketoglutarate, which may induce significant changes in the mitochondrial energy metabolism and cellular bioenergetics required for the growth and proliferation of EBV-associated cancer cells. Here, GLS is linked to nasopharyngeal carcinoma.