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Abstract Background & aim: Aspartame is an artificial sweetener that has been used extensively in over 200 million people in more than 90 countries in various food products and pharmaceuticals. There are many controversial reports about the toxicity of aspartame on various tissues of the body. Therefore, the aim of the present study was to determine the effect of long-term aspartame on histomorphometric and histochemical adrenal glands in NMRI mice. Methods: In the present experimental study, 36 male NMRI male rats weighing 20-25grs were perched from Pasteur Institute of Experimental Animals. The rats were randomly divided into four groups: three groups received aspartate at dose of 40 (low dose of aspartame), 80 (medium dose of aspartame) and 160 (high dose of aspartame) mg/kg body weight by oral gavage for 90 days, respectively. And the control group was also considered. 24 hours after the last treatment, histological and histomorphometric changes were evaluated by digital microscopy. Also, specific stains of periodic acid schiff, trichrome masson and toluidine blue were used to determine carbohydrate compounds, amount of fibrosis and the number of mast cells in adrenocortical tissue. The collected data were analyzed by one-way ANOVA and Tukey's test. Results: Aspartate in the high dose group resulted in the collusion of cellular structure between glomerular cell and reticularis cells. Also, aspartame in the fashiculate region in medium to high dose groups caused cellular collusion and disintegration of the cellular columns and inflammatory and necrosis coronals were also observed in the feccilata region. In the central part of the adrenal gland, there were medium and high necrosis points in the adrenal gland. Histometric changes showed a significant increase in the size of the sponge cells, the number of mast cells, and the thickness of the layers of the fashiculate and reticularis. A significant reduction was observed in the large diameter of the central region of the adrenal gland in medium and high dose groups. No significant changes were seen in glomerular layer thickness and adrenal gland capsule parameters. In the histochemical studies of Trichromus Mason staining, it was found that aspartame increased the fibrosis tissue in the high dose group. Also, there was no significant change in the level of carbohydrate in the periodic acid staining in the groups. Conclusion: The results of this study revealed that aspartame as an oxidant in medium and high dose groups resulted in negative effects on histomorfrometry parameters and tissue damage in the adrenal gland by producing reactive oxygen species. It also increased the number of mast cells and fibrosis of the adrenocortical gland tissue.