Glutamine serves as a major fuel source for tumor cell proliferation while simultaneously playing an essential role in maintaining gastrointestinal health and immune function. Controversy exists regarding glutamine supplementation for cancer patients undergoing chemotherapy and radiation, with concerns that it may stimulate cancer growth. The present study is the first to examine the effects of dietary glutamine supplementation (0.4g/kg/day) on the growth of malignant gliomas, which utilize large amounts of glutamine to satisfy metabolic demands. Brain bioluminescence and subcutaneous tumor volumes were used to assess the influence of glutamine supplementation on the growth of the syngeneic VM-M3 and CT-2A preclinical models of glioma. Glutamine supplementation had no significant effect on the orthotopic growth of the VM-M3 or the CT-2A gliomas when compared to non-supplemented controls. However, glutamine supplementation significantly increased tumor volume by 28% in the VM-M3 and by 166% in the CT-2A tumors when grown subcutaneously outside the central nervous system (CNS) relative to controls. Additionally, glutamine supplementation increased serum glutamine despite a localized decrease in intratumoral glutamine concentrations. Caution is warranted when considering glutamine supplementation in patients with glutamine-dependent malignancies. Further studies are needed to better understand the potential risks and benefits of glutamine supplementation in cancer therapy.