10 male KM mice were divided into normal group and Dendrobium officinale polysaccharides(DOP) treated group. Mice in the treated group were gavaged with DOP at the clinical equivalent dose(350 mg/kg) for 8 weeks, once a day, 0.4 mL each rat at a time, and the control group was gavaged with sterile water. To investigate the effect of DOP on gut microbiota of mice, the intestinal contents and mucosa were collected for 16S rRNA gene sequencing and function prediction. The results showed that DOP increased α diversity of lumen-associated microbiota, especially mucosa-associated microbiota, but there was no significant difference in β diversity. DOP significantly increased the abundance of Diaphorobacter in intestinal lumen and Muribaculaceae, Staphylococcus in intestinal mucosa. The relative abundance of short-chain fatty acids producing bacteria including Akkermansia, Phascolarctobacterium, Turicibacter, Faecalibacteriu, Parabacteroides, Parasutterella and beneficial bacteria such as Candidatus_Arthromitus, Bifidobacterium and Prevotella_9 were increased. In addition, DOP increased metabolism pathway of cofactors, vitamins and amino acid in intestinal lumen and mucosa.