Glioma is the most common type of brain tumor, and has the worst prognosis in human malignancy. Experimental evidence suggests that the use of high concentrations of various amino acids may perturb neoplastic cell growth. Thus, the aim of this study was to investigate whether essential amino acids can alter the growth and proliferation of glioma cells. Studies were performed using C6 rat glioma cell lines. High concentration of L-leucine induced growth arrest of glioma cell lines. Terminal transferase uridyl nick end labeling assay and cell cycle analysis showed that the effect of L-leucine on glioma cells growth was not cytotoxic, but rather cytostatic. Additionally, the extracellular signal-regulated protein kinase was activated in L-leucine-treated glioma cells, and inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK) enhanced the effect of L-leucine on glioma cell growth. These data suggest that high concentration L-leucine combined with inhibition of MEK is a potential strategy for glioma cell growth arrest.