Abstract Lentinan (LNT) is a polysaccharide from Shiitake mushrooms that has significant effects regarding antioxidant, hypoglycemic, anti-inflammatory activities, etc., and this effect is influenced by the human intestinal flora. However, the interactions between LNT and human intestinal microorganisms, and their metabolic processes, remain unclear. In this study, the fermentation characteristics of LNT and its effects on intestinal flora and metabolites were studied by in vitro fermentation. Our results indicated that LNT was effectively utilised by intestinal microbiota, and the utilisation rate was 62.7 ± 3.0% after fermentation for 48 hr. The concentrations of short-chain fatty acids (mainly acetic acid and propionic acid) were increased to 4.157 ± 0.197 and 5.847 ± 0.283 mM, respectively. LNT also increased the relative abundance of beneficial bacteria, such as Bifidobacterium and Parabacteroides, while reducing harmful bacteria like Veillonella and Klebsiella. Metabolomics and microbial correlation analysis revealed that the catabolism of LNT was also accompanied by amino acid catabolism, especially alanine, aspartic acid, glutamate, and the biosynthesis of adenosine 5’-monophosphate, etc. These findings suggest that LNT can support gut health and demonstrates potential as a prebiotic.