In this study we evaluated the impact of juglone on rat glioma C6 cell culture viability, proliferation and invasiveness in vitro. Juglone induced C6 cell death with EC50 concentrations equal to 10.4 ± 1.6 µM after 24h incubation. At relatively low concentrations juglone significantly decreased cell proliferation, reduced spheroid invasiveness and suppressed “wound” healing. In addition, generation of intracellular reactive oxygen and nitrogen species (RS) was detected in cells treated with juglone. Noteworthy, juglone was relatively stable in cell culture medium and levels of H2O2 generated from juglone due to its probable reaction with medium components were not sufficient to affect the viability of glioma cells. Moreover, addition of catalase to the cell medium did not reduce the cytotoxicity of juglone. Therefore, we propose that cell death may be induced through the action of RS other than H2O2, However the direct effect of juglone on the cellular targets could not be excluded either. In conclusion, juglone exerted cytotoxic, anti-proliferative and anti-invasive effects on C6 rat glioma cells in vitro.