BACKGROUND: One of the most artificial sweeteners is Aspartame which is commonly  used in a variety of foodstuffs. Objectives: This study has been carried out in order to evaluate the effects of Aspartame on histology and histometry of duodenum. Methods: Forty female Balb/C mice, 21 days of age were selected and initial weight was determined. The treatment groups received 100, 200 and 400 mg/kg of Aspartame for 6 weeks, control group received only distilled water. At the end of experiment, the mice were reweighed. Then the duodenal tissue sections were prepared and stained with H&amp;E. Besides the histological study, histometric data were collected by a light microscope equipped with Axiovision software. Results: The body weights in treatment groups (100, 200 and 400 mg/kg) were 5.57±1.18, 4.47±0.89 and 5.84±0.57 respectively, which in comparison with the control group (9.38±0.81) showed a significant difference (p<0.05). The histological study showed that the rate of destruction in the cells and mucosal structures, at the dose of 200 mg/kg compared to the dose of 100 and 400 mg/kg has been increased. In histometric aspect, abundance of duodenal villi, height of the villi and thickness of duodenal mucosa, only in the experimental group of 200 mg/kg were significantly increased compared to the control group (p<0.05). Whereas the width of villi (width of the apex, body and the base), in all of the experimental groups showed a significant decrease compared to the control group (p<0.05). The thickness of musculature of duodenum in experimental groups had no significant differences with the control group. Conclusions: Based on this study, it can be concluded that Aspartame can cause some histological and histometrical changes in duodenum.