In this study, the in vitro digestion and fermentation of two intra-polysaccharide fractions (IPS1 and IPS2) from Paecilomyces cicadae TJJ1213 were investigated. The constituent monosaccharides of IPS1 and IPS2 were not changed after simulated saliva, gastric and small intestinal digestion. However, they can be hydrolyzed and utilized by gut microbiota, and short-chain fatty acids (SCFAs) level were increased after IPS1and IPS2 treatments. Furthermore, 16S rRNA sequencing analysis of fermentation samples were performed. Alpha-diversity, beta-diversity and taxonomic composition differences analysis revealed that IPS1 and IPS2 promoted the proliferation of beneficial bacteria and modulated the overall structure of gut microbiota. Taxonomic comparison analysis found that IPS1 increased the relative abundances of beneficial bacteria including Megamonas, Bifidobacterium and Lactobacillus, while IPS2 could increase the abundance of Bacteroides, Parabacteroides and Phascolarctobacterium. In addition, they can also decrease the levels of pathogenic bacteria containing Escherichia-Shigella, Klebsiella and Fusobacterium. These results indicated that IPS from Paecilomyces cicadae TJJ1213 could be used as potential candidates for new functional foods.