Abstract: An in vitro simulation of digestion and fermentation model with feces bacteria was used to explore the probiotic capacity of Polyonatum cyrtonema Hua. polysaccharide (PCP) by 16S rDNA gene sequencing and determining changes in short-chain fatty acids via GC-MS. The results showed that the digestibility of PCP was not obviously found by in vitro simulating saliva-gastrointestinal digestion model. The addition of PCP could rapidly reduce the pH of fermentation broth. PCoA analysis showed that PCP had the effect of altering gut microbiota, which was similar with inulin. At the phylum level, compared to the blank control group, the relative abundance of Firmicutes and Actinomycetes was significantly increased (P<0.05) in the PCP group, while that of Proteobacteria was significantly decreased (P<0.05). At the genus level, the growth of beneficial bacteria Bifidobacterium and Megamonas was significantly promoted (P<0.05), while the growth of opportunistic bacteria Enterobacter and Escherichia-Shigella was significantly inhibited (P<0.05). Moreover, PCP greatly increased the contents of short-chain fatty acids, among which the contents of acetic acid and propionic acid were significantly increased (P<0.05). The present results suggest that PCP may enhance the production of short-chain fatty acids via gut microbiome regulation.