Abstract: Objective: To explore the changes of active components in Polygonatum sibiricum extract and its regulation on intestinal flora by in vitro simulated digestion combined with 16S rDNA high-throughput sequencing technology. Methods: P. sibiricum extract was prepared by heating reflux method. The contents of polysaccharides, saponins and flavonoids in P. sibiricum extract before and after simulated digestion in vitro were determined, and its inhibitory ability on α-glucosidase was investigated. The effect of P. sibiricum extract on intestinal flora was analyzed based on 16S rDNA high-throughput sequencing. Results: The P. sibiricum extract contained polysaccharides, saponins and flavonoids, and the contents were 0.4732, 0.0632 and 0.0754 mg/mL, respectively. After in vitro simulated digestion, the contents of polysaccharides, saponins and flavonoids were significantly reduced (P<0.05), and the addition of digestive enzymes was significantly lower than that of the group without digestive enzymes (P<0.05). In addition, the inhibitory ability of P. sibiricum extract on α-glucosidase was significantly reduced after digestion (P<0.05). With the increase of in vitro fermentation time, the pH of the fecal fermentation broth in the P. sibiricum extract treatment group decreased from the initial 6.85 to 4.13, and the fermentation broth was weakly acidic after 48 h. The analysis of intestinal flora showed that P. sibiricum extract could change the structure of intestinal flora, especially at the level of phylum and genus. P. sibiricum extract increased the abundance of Firmicutes, decreased the abundance of Proteobacteria, increased the relative abundance of Bifidobacterium, and significantly decreased the relative abundance of Fusicatenibacter (P<0.05). Conclusion: The contents of polysaccharides, saponins and flavonoids in P. sibiricum extract were significantly decreased after in vitro simulated digestion (P<0.05), and the inhibitory effect on α-glucosidase was decreased. It could change the structure of intestinal microbial community and had a certain regulatory effect on intestinal flora. This study can provide a theoretical reference for the deep processing of P. sibiricum and the targeted regulation of intestinal flora.