More than 90 countries have given the artificial sweetener aspartame the green light to be used in thousands of food and beverage products. The artificial dipeptide sweetener aspartame [APM; L- aspartyl-L- phenylalanine methyl ester] is present in many products especially unsweetened and sugar products. These products are frequently utilized by people trying to lose weight or patients with diabetes. Concern relating to the possible adverse effect has been raised due to aspartames metabolic components. Aspartame is rapidly and completely metabolized in humans and experimental animals to aspartic acid (40%), phenylalanine (50%) and methanol (10%). Methanol, a toxic metabolite is primarily metabolized by oxidation to formaldehyde and then to formate these processes are accompanied by the formation of superoxide anion and hydrogen peroxide. This study focus is to understand whether the oral administration of aspartame (40 mg/kg b.w.) for 90 days, have any effect on membrane bound ATPase’s, antioxidant status and immune response (cell and humoral) of rats. To mimic human methanol metabolism, folate deficient rats were used. After 90 days of aspartame administration, shows free radical production by a significant increase in LPO and nitric oxide (NO) level and decrease in both enzymatic and nonenzymatic antioxidant level which alters the immune response. This study concludes that oral administration of aspartame (40mg/kg b.w) for longer duration may cause oxidative stress on immune organs and altered the immune response (cell and humoral) in wistar albino rats.