© 2021 by Begell House, Inc.In this study, we hypothesize that Cantharellus cibarius polysaccharide (CCP) could contribute to the growth performance, specific growth rate, and bacteriological, biochemical, and histological aspects of the gut in rats. Three groups of seven rats received one of three diets; negative control, treatment, and positive control groups were supplemented with commercial feed, + 0.25% CCP, and + 0.25% inulin, respectively. The highest Lactobacillus and the lowest Clostridium counts (1.84 × 109 and 0.03 × 109 CFU/ml, respectively) were obtained in the colon content of rats fed with CCP. The prebiotic index value of the treatment group (9.39) was higher than that of the positive control, inulin (7.74). There was a slight increase in total cholesterol and low-density lipoprotein and a slight decrease in high-density lipoprotein and triglyceride levels in the treatment group. Histologically, any important increase in mesenteric mast cell counts and degranulation has not been determined. The results indicate that CCP displays a prebiotic effect through the change of the intestinal microbiota. To our knowledge, the pre-biotic effect of polysaccharide of C. cibarius fruiting body under in vivo conditions is reported here for the first time.