Background: Glioblastoma multiforme (GBM) is the most fatal glioma with poor prognosis. C6 glioma is an experimental model to simulate GBM growth and biology. GBM benefits from Warburg effect (aerobic glycolysis ending in lactate formation even in the presence of oxygen) in tumor growth, invasion and metastasis. Citric acid is an antioxidant and inhibitor of glycolysis pathway (phosphofructokinase inhibitor) that constitutes the major source of energy supply to aggressive cancer cells. Citrate is a promising inhibitor of Warburg effect through blocking glycolysis upstreatm of lactate formation step. Methods: Ability of citric acid to inhibit experimental GBM colony formation and anchorage-independent growth were investigated. Results: Citrate induced a dose-dependent inhibition of growth and proliferation of glioma colonies (attached to substratum). High citrate dose (9 mM) inhibited initial formation of glioma colonies. A similar picture was observed where citrate induced a dose-dependent inhibition of growth of glioma colonies in soft agar (i.e. not attached to a substratum). High citrate dose (9 mM) inhibited initial formation of glioma colonies. In conclusion, citrate inhibited 3D tumor models of GBM. Citric acid inhibited clonogenic power of glioma cells. Conclusion: Citric acid may be a promising therapeutic modality for glioma and glioblastoma. That is quite promising in treating GBM tumors and can be generalized for research in different tumors.