Glutamine is a versatile nutrient essential for cancer cell survival, growth, and metabolism. It plays a critical role in energy production, redox balance, macromolecular synthesis, and signaling, making it a promising target for cancer diagnosis, monitoring, and treatment. This review discusses the metabolic functions of glutamine in cancer, its role in supporting cancer hallmarks, and recent advances in imaging and therapeutic strategies targeting glutamine metabolism. Glutamine is the most abundant amino acid in plasma and is essential for many cancer cells, especially those with oncogene-dependent glutamine addiction. It is metabolized through enzymes like glutaminases (GLSs), which convert glutamine to glutamate, a precursor for glutathione and other metabolites. Glutamine-derived α-ketoglutarate is used in the TCA cycle and by dioxygenases involved in protein and DNA modification. In cancer cells with impaired mitochondrial function, glutamine is reductively carboxylated to support TCA cycle intermediates. Glutamine supports cancer hallmarks such as energy formation, proliferative signaling, replicative immortality, and resistance to cell death. It modulates pathways like mTOR and hexosamine synthesis, which are crucial for cell growth and survival. Malic enzymes, which produce NADPH, are involved in glutamine metabolism and can influence senescence and tumor growth. Organ-specific glutamine metabolism varies, with some organs acting as net producers and others as consumers. Cancer alters inter-organ glutamine trafficking, affecting tumor growth and host metabolism. Glutamine is used by tumors and cancer-associated immune cells, and its metabolism can be targeted for therapy. Tumors differ in their dependence on glutamine, with some cells being highly dependent and others independent. This variability highlights the need for personalized approaches to predict tumor response to glutamine metabolism inhibitors. Imaging techniques, such as PET using glutamine analogs, and hyperpolarized nuclear magnetic resonance can provide insights into glutamine metabolism in tumors. Pharmacological strategies to inhibit glutamine metabolism include inhibitors of GLS, such as compound 968 and BPTES, and amino acid analogs like acivicin and azaserine. These agents target specific metabolic nodes and show promise in preclinical models. The development of safe, potent inhibitors of key metabolic enzymes could lead to effective therapeutic regimens for cancer. In conclusion, glutamine metabolism is a critical aspect of cancer biology, offering opportunities for diagnostic and therapeutic interventions. Advances in imaging and targeted therapies are paving the way for more effective cancer treatment strategies.Glutamine is a versatile nutrient essential for cancer cell survival, growth, and metabolism. It plays a critical role in energy production, redox balance, macromolecular synthesis, and signaling, making it a promising target for cancer diagnosis, monitoring, and treatment. This review discusses the metabolic functions of glutamine in cancer, its role in supporting cancer hallmarks, and recent advances in imaging and therapeutic strategies targeting glutamine metabolism. Glutamine is the most abundant amino acid in plasma and is essential for many cancer cells, especially those with oncogene-dependent glutamine addiction. It is metabolized through enzymes like glutaminases (GLSs), which convert glutamine to glutamate, a precursor for glutathione and other metabolites. Glutamine-derived α-ketoglutarate is used in the TCA cycle and by dioxygenases involved in protein and DNA modification. In cancer cells with impaired mitochondrial function, glutamine is reductively carboxylated to support TCA cycle intermediates. Glutamine supports cancer hallmarks such as energy formation, proliferative signaling, replicative immortality, and resistance to cell death. It modulates pathways like mTOR and hexosamine synthesis, which are crucial for cell growth and survival. Malic enzymes, which produce NADPH, are involved in glutamine metabolism and can influence senescence and tumor growth. Organ-specific glutamine metabolism varies, with some organs acting as net producers and others as consumers. Cancer alters inter-organ glutamine trafficking, affecting tumor growth and host metabolism. Glutamine is used by tumors and cancer-associated immune cells, and its metabolism can be targeted for therapy. Tumors differ in their dependence on glutamine, with some cells being highly dependent and others independent. This variability highlights the need for personalized approaches to predict tumor response to glutamine metabolism inhibitors. Imaging techniques, such as PET using glutamine analogs, and hyperpolarized nuclear magnetic resonance can provide insights into glutamine metabolism in tumors. Pharmacological strategies to inhibit glutamine metabolism include inhibitors of GLS, such as compound 968 and BPTES, and amino acid analogs like acivicin and azaserine. These agents target specific metabolic nodes and show promise in preclinical models. The development of safe, potent inhibitors of key metabolic enzymes could lead to effective therapeutic regimens for cancer. In conclusion, glutamine metabolism is a critical aspect of cancer biology, offering opportunities for diagnostic and therapeutic interventions. Advances in imaging and targeted therapies are paving the way for more effective cancer treatment strategies.