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Coix seed polysaccharides had received increasing attention due to their diverse biological activities. In this study, a homogeneous polysaccharide (CSPW) was extracted and purified from coix seed. Furthermore, the saliva-gastrointestinal digestion and fecal fermentation behavior of CSPW were simulated in vitro. The results showed that CSPW was mainly composed of glucose. It cannot be degraded by the simulated salivary and intestinal digestive system, but can be degraded by the simulated gastric digestive system. After fermentation for 24 h, CSPW promoted the production of short-chain fatty acids (SCFAs), with acetic acid, propionic acid and n-butyric acid being the main metabolites. In addition, CSPW could significantly regulate the composition and microbial diversity of gut microbiota by increasing the relative abundance of beneficial bacteria, such as Limosilicactobacillus, Bifidobacterium and Collinsella. Finally, further analysis of functional prediction revealed that amino acid metabolism, nucleotide metabolism and carbohydrate metabolism were the most important pathways for CSPW to promote health. In summary, our findings suggested that CSPW could potentially be used as a good source of prebiotics because it can be used by gut microbiota to produce SCFAs and regulate the gut microbiota. • CSPW was a homogeneous polysaccharide from coix seed and can be consumed by gut microbiota. • CSPW was beneficial for the growth of beneficial bacteria and inhibited harmful microbiota. • The gut microbiota produced more beneficial metabolites by CSPW modulated. • CSPW showed the potential of prebiotics and had beneficial effects on the host. [ABSTRACT FROM AUTHOR]