Grifola frondosa polysaccharide (GFP) as the natural compounds have been reported to exert diverse bioactivities. The aim of this study was to investigate the regulatory effects of GFP on intestinal microbiota in type 2 diabetic mice. The changes of microbiota in faeces of the diabetic mice upon high fat diet were determined and the V3 region of the 16S rRNA was sequenced by Illumina MiSeq high-throughput sequencing platform. Eighty operational taxonomic unit were identified to be shared by all samples, and the quality and richness of sequencing were assessed via rarefaction and rank abundance curves. The diversity of gut flora was slightly improved in diabetic mice after GFP treatment. The composition and relative abundance of gut microbiota at phylum and genus levels were altered by GFP. The abundance of Robinsoniella, Flavonifractor, Anaerotruncus, and Desulfvibrio were found to concentrate on diabetic mice through LEfSe analysis. Furthermore, Alloprevotella and Burkholderia had strong relevancy with other gut flora. Based on the findings, GFP might be a desired candidate on ameliorating the intestinal unbalance in diabetes.