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Polysaccharides are biological macromolecules that are difficult to absorb into intestinal epithelial cells for exerting activities, whereas the interaction between polysaccharides and gut microbiota might be an alternative method. This study aimed to explore the in vitro digestion of hawthorn polysaccharides (HPS) and their interaction with the gut microbiota. Results showed that the content of reducing sugars increased slightly during gastric digestion. However, no free monosaccharide was detected during the whole simulated digestion process, indicating that HPS was indigestible. The total carbohydrate residue decreased during in vitro fermentation. This result was due to the utilization by the gut microbiota. Meanwhile, short-chain fatty acids were produced due to the utilization of HPS. Notably, HPS could significantly modulate the composition of human gut microbiota; in particular, the relative abundances of Megasphaera, Acidaminococcus and Mitsuokella increased, whereas the relative abundances of Escherichia Shigella and Fusobacterium decreased. It was suggested that HPS could decrease the abundances of harmful intestinal microbiota and regulate the proportion of beneficial bacteria in the intestinal tract. Overall, the beneficial effects of HPS were believed to be related to the gut microbiota and could be used as a potential dietary supplement.