Abstract The use of the artificial sweetener aspartame has long been contemplated and studied by researcher around the world regarding their varying negative effects. The present study aims to evaluate the long-term effect of aspartame (75mg/kg) on liver and brain antioxidant status with histopathological changes in liver and renal cortex in Wistar strain albino rats. Many existing reports, which are available, state that aspartame releases toxic metabolites during metabolism, in which methanol is considered to be one. To mimic the human methanol metabolism, methotrexate (MTX) treated rats were included to study the aspartame effects. There were significant decrease in reduced glutathione (GSH), glutathione reductase (GR) along with marked increase in lipid peroxidation (LPO), glutathione-S-transfrease (GST), γ-glutamyl transpeptidase (γ-GT), protein carbonyl and formate level, indicating changes in the antioxidant status of liver and brain. There were also significant histological changes in the liver and renal cortex. Hence, methanol per se and its metabolites may be responsible for the antioxidant status and histological changes in liver and renal cortex. Hence, it can be concluded that long-term aspartame may be responsible for oxidative stress and the hepato-renal toxicity.