Introduction: Astrocyte, S100B and nitric oxide may have a role in the pathogenesis and treatment of epilepsy. However, the effects of nitric oxide and S100B on the gliotoxic effects of chemical convulsants such as pentylenetetrazole (PTZ) is unknown. Therefore, we aimed to evaluate the effects of S100B and nitric oxide on gliotoxicity of PTZ in a 1321NI1 astrocytic culture. Methods: The 1321N1 astrocytes were exposed to PTZ (40mM), arundic acid (50μM) or both of them for 24h. In addition, we poured L-arginine (100 or 500μM), N-nitro-L-arginine methyl ester (100 or 500μM), 7-nitroindazole (30 or 100μM) and aminoguanidine (50 or 100μM) to the culture media contained PTZ, arundic acid or both of them and incubated for 24h. Cell viability was measured by the methylthiazolyldiphenyl-tetrazolium bromide reagent and the S100B protein level was measured using an enzyme-linked immunosorbent assay. Results: There was a negative correlation between cell viability in astrocytes and the intracellular S100B levels. PTZ decreased cell viability, but it increased the intracellular S100B levels. Arundic acid, N-nitroarginine methyl ester, 7-nitroindazole and aminoguanidine reversed the PTZ effects on cell viability and intracellular S100B levels. Adding the L-arginine to PTZ plus arundic acid reduced the modulatory effects of arundic acid on PTZ. Conclusion: Nitric oxide and S100B have a role in gliotoxicity of PTZ in cell culture. Arundic acid suppresses PTZ-induced S100B elevation and gliotoxicity possibly by modulation of the nitric oxide pathway.