Glutaminase catalyzes the conversion of glutamine to glutamate, the first and rate-limiting step of glutaminolysis. Both kidney-type glutaminase (GLS1) and liver-type glutaminase (GLS2) are found in mammals. GLS1-mediated glutathione synthesis plays an essential role in redox homeostasis and contributes to increased survival of postimplantation bone cells preconditioned to the hypoxic and ischemic environment in the bone defect site. In addition, KEAP1–NRF2-mutant LUAD (KRAS-mutant lung adenocarcinoma) tumors are dependent on increased glutaminolysis. Furthermore, recent studies showed higher glutaminolysis and glucose production from glutamine in human primary hepatocytes with GLS2 gain-of-function missense mutations. These findings suggest GLS1 and GLS2 as potential targets in the therapy of bone regeneration and in the treatments of diseases such as cancer and hyperglycemia, respectively.