Abstract: To investigate the intestinal fermentation characteristics of different endopolysaccharides (MPS) sub-fractions MPS-1, MPS-2, MPS-3, and MPS-4 from Monascus purpureus mycelium, an in vitro pig colon model was used to determine the changes in molecular weight of the polysaccharides, the contents of short-chain fatty acids (SCFAs), and gut microbiota at different fermentation times. The results showed that after intestinal fermentation, the acetic acid, propionic acid, and butyric acid production in each MPS treatment group was higher. At 48 h of fermentation, the total SCFAs content produced by the MPS-2 and MPS-3 treatment groups increased significantly (P<0.05), from 22.80 and 17.20 mmol/L to 63.08 and 71.61 mmol/L, respectively. The total SCFAs concentration of each polysaccharide treatment group was MPS-3>MPS-2>MPS-1>MPS-4 in descending order. The 16S rDNA results revealed that after MPS treatment, Firmicutes and Bacteroidetes were the main dominant bacteria at the phylum level, and the ratio of Firmicutes/Bacteroidetes was reduced. At the genus level, with the extension of fermentation time, the abundance of beneficial bacteria such as Bacteroides, Prevotella, and Parabacteroides increased, while the abundance of harmful genera, including Streptococcus and Clostridium, decreased significantly (P<0.05). Among them, the MPS-2 and MPS-3 treatment group had the higher abundance of beneficial bacteria and the lower abundance of harmful bacteria. In summary, MPS have great potential as prebiotics in the fields of functional foods and biomedical applications.