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The present study aimed to investigate the modulatory effect of polysaccharide from Artocarpus heterophyllus Lam. pulp (JFP-Ps) on the diversity and structure of gut microbiota in mice. 24 male Kunming mice were randomly divided into four groups: Control group, JFP-Ps (50 mg/kg BW) group, JFP-Ps (100 mg/kg BW) group and JFP-Ps (200 mg/kg BW) group. The colonic feces of mice were collected after treating with JFP-Ps in different doses for 2 weeks. Then the DNA was extracted and the V3~V4 region of the 16S rRNA gene of the intestinal flora was amplified. The regulatory effect of JFP-Ps on the gut microbiota of mice was studied using Illumina Miseq high-throughput sequencing technology. The results showed that Bacteroidetes and Firmicutes were the dominant intestinal flora, and JFP-Ps modulated the composition of Bacteroidetes and Firmicutes in comparison with the control group. At the genus level, the abundance of SCFAs-producing bacteria, including Bacteroides, Allobaculum, Lactobacillus and Parabacteroides were significantly increased in response to JFP-Ps treatment, but a significant decrease in Prevotella. These results indicated that JFP-Ps could improve the diversity of gut microbiota by regulating the composition and structure of dominant bacteria such as Bacteroidetes and Firmicutes. This study would provide a theoretical basis for JFP-Ps in intestinal prebiotics.