This research was aimed to construct the nanoparticles based on Hohenbuehelia serotina polysaccharides for encapsulation of quercetin (QC-HSP NPs), and investigate their effects on intestinal function and gut microbiota in mice. Results showed that in comparison with HSP and control, QC-HSP NPs significantly improved immune organ indexes, colon length, fecal moisture content and intestinal peristalsis capacity of mice. The productions of short-chain fatty acids (SCFAs) in colon were also increased after treatment with QC-HSP NPs, while the colonic fecal pH was decreased and defecation time was shortened. Through analysis of 16S rRNA sequencing, QC-HSP NPs could increase α and β diversities of gut microbiota, modulate their structure and composition, and increase the relative abundance of beneficial bacteria together with reducing the richness of harmful bacteria. In addition, QC-HSP NPs ameliorated the metabolic functions of gut microbiota by modulating metabolic pathways. This study suggested that QC-HSP NPs might be served as a prebiotic for protecting intestinal health. • Quercetin-loaded nanoparticles based on Hohenbuehelia serotina polysaccharides were prepared. • QC-HSP NPs could be utilized by gut microbiota to produce more SCFAs. • The intestinal peristalsis, fecal moisture and defecation time were improved by treatment with QC-HSP NPs. • The structure and composition of gut microbiota in mice were modulated by treatment with QC-HSP NPs. [ABSTRACT FROM AUTHOR]