Clear cell renal cell carcinomas (ccRCC) are largely driven by HIF2α and are avid consumers of glutamine. However, inhibitors of glutaminase1 (GLS1), the first step in glutaminolysis, have not shown benefit in phase III trials, and HIF2α inhibition, recently FDA-approved for treatment of ccRCC, shows great but incomplete benefits, underscoring the need to better understand the roles of glutamine and HIF2α in ccRCC. Here, we report that glutamine deprivation rapidly redistributes GLS1 into isolated clusters within mitochondria across diverse cell types, excluding ccRCC. GLS1 clustering is rapid (1-3 hours) and reversible, is specifically driven by the level of intracellular glutamate, and is mediated by mitochondrial fission. Clustered GLS1 has markedly enhanced glutaminase activity and promotes cell death under glutamine-deprived conditions. We further show that HIF2α prevents GLS1 clustering, independently of its transcriptional activity, thereby protecting ccRCC cells from cell death induced by glutamine deprivation. Reversing this protection, by genetic expression of GLS1 mutants that constitutively cluster, enhances ccRCC cell death in culture and suppresses ccRCC growth in vivo . These finding provide multiple insights into cellular glutamine handling, including a novel metabolic pathway by which HIF2α promotes ccRCC, and reveals a potential therapeutic avenue to synergize with HIF2α inhibition in the treatment of ccRCC.