Paracetamol is an active metabolite with analgesic and antipyretic properties of phenacetin, which is sold without a prescription in our country and in many countries. However, the effect of paracetamol on oxidative stress due to glutamate-induced cytotoxicity remains unclear. This study aims to investigate the effect of an appropriate dose of paracetamol on nitric oxide and increased oxidative stress as a result of glutamate-induced cytotoxicity in C6 cells. The cells were divided into 4 groups as Control group, Glutamate group, Paracetamol group, and Paracetamol+Glutamate group. Cell viability rate between groups was measured by XTT assay. Oxidative stress and antioxidant levels were measured with TOS and TAS elisa kits. Paracetamol at all concentrations significantly increased cell viability in C6 cells (p < 0.001). Paracetamol also increased TAS levels (p <0.01) while significantly decreased TOS levels (p < 0.001). In addition, paracetamol was observed to decrease TNF-α and NO levels (p <0.001). In conclusion, paracetamol has protective feature on glutamate-induced cytotoxicity in C6 glial cells by suppressing oxidative stress. The results of this study show that when the appropriate dose of paracetamol is used, it can be a crucial promoter agent in glutamate toxicity-induced neurodegeneration.