Polysaccharide is the major biologically active constituent in functional mushroom Ganoderma lucidum. This study was aim to investigate the utilizability of G. lucidum polysaccharide by human fecal microbiota and its prebiotic potential based on the Simulator of Human Intestinal Microbial Ecosystem (SHIME). GLP was obtained from fruiting bodies of G. lucidum with average molecular weight of 133.1 KDa. Short-term colonic fermentation of GLP revealed the continuously decreased contents of total carbohydrates and reducing sugar as well as pH. Gas chromatography analysis indicated that GLP promoted the production of acetic, propionic and butyric acids. The 16S rDNA amplicon sequencing analysis showed that GLP improved the microbial diversity and ratio of Bacteroides to Firmicutes., and sharply enriched Bacteroides ovatus and B. uniformis. Therefore, GLP can be utilized by human fecal microbiota and then modify microbial community and metabolic function, and may serve as novel prebiotics to bring health benefits for human.