Background: Recently, exploring novel dietary nondigestible carbohydrates, which are able to influence the gut flora, has drawn much attention. The objective of this study was to find out the effective dose of levan, as a prebiotic, in rats in order to further apply in food industry. Methods: Levan at various doses (2-10%) was orally administered to male Wistar Albino rats once a day for 90 days. At the end of experiment, fecal and blood samples were collected to measure gut bacteria population and to carry out serum biochemical assay. The rats were sacrificed, and the colon tissues were stained with H&E and analyzed by histopathology. Results: Of note, levan effectively controlled body weight gain in the rats. Serum biochemical analysis revealed that 5% levan significantly diminished the serum level of total cholesterol, LDL, and glucose as well. More notably, 5% levan intake significantly increased the abundance of BIFIDOBACTERIA population, highlighting its bifidogenic effect. Furthermore, our histopathological result revealed that daily intake of levan was associated with a higher degree of thickness of the mucosa layer compared to the rats in control group. Moreover, these findings manifested no colon inflammation in the rats fed with levan. Conclusion: The findings of this study provide the fundamental data to use levan at a definite dose for further development in functional foods.