This study was conducted to determine the protective role of ascorbic acid (AA) against cytotoxicity of the phenthoate (PTA) on hepatocytes. Lipid peroxidation (LPO), nitric oxide (NO), antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase, gamma glutamyl transferase and glutathione-S-transferase activities were measured. Non-enzymes antioxidant levels of reduced glutathione (GSH), ferric reducing antioxidant power (FRAP), AA and total proteins (TP) were determined. The results were demonstrated that the 2 hr-LC50 obtained for PTA value was 79.94 mM. LPO and NO were increased in hepatocytes-treated with PTA. PTA-induced oxidative stress in the isolated hepatocytes by decreasing the levels of some antioxidant enzymes. GSH levels were also diminished in PTA treated-hepatocytes as compared to DMSO hepatocytes. FRAP, AA and total protein were also decreased following treatment with PTA. These findings suggest that cytotoxicity of PTA is mediated by increasing free radical formation and decreasing the antioxidants. PTA hepatotoxicity increased in a time- and concentration-dependent manner. The AA can be very effective in perhaps reducing the extent of injury and in overcoming oxidant damage caused by PTA.