Abstract: In this study, the potential synergistic relationship between reduced glutathione and vitamin E on ethanol toxicity in isolated rat hepatocytes were examined. It was observed that treatment of ethanol (5-25 mM) caused decrease in cell viability but in presence of Rotenone, cell viability decreased to a greater extent. Inhibitory concentration (IC50) was found to be 20 mM of ethanol treated with rotenone (10 μM) for 2 h. Treatment of ethanol with rotenone (20 mM±10 μM) caused increase in lipid peroxidation, increase of lactate dehydrogenase and decrease in reduced glutathione content. Reduced glutathione and α-tocopherol, alone and in combination, were added to hepatocyte suspension in an attempt to protect cells against alcohol induced cell damage. Pretreatment with reduced glutathione and vitamin E independently, increased the cell viability to 78.97 and 70.42%, respectively, but a combination of reduced glutathione and vitamin E increased cell viability to 92.12%. Vitamin E reduced lipid peroxidation by 41.32% but in combination with reduced glutathione decreased lipid peroxidation by 61.15%. The results suggest an interaction between GSH and Vitamin E in protecting ethanol induced hepatocytes damage.