Ganoderma lucidum and Poria cocos are the medicinal mushrooms that have been used as traditional Chinese medicines and consumed as functional foods. We hypothesized that these mushroom polysaccharides could contribute to health benefits, in part, by modulating gut microbiota (GM). In this study, mushroom polysaccharides derived from fruiting bodies and mycelia were administrated daily (750 mg/kg) to 6 weeks-old C57BL/6J mice for 15 days. Then, mice GM was evaluated using ERIC-PCR and 16S amplicon sequencing. A total of 2.59 million high-quality reads (>200 bp) were processed and evaluated for GM profiling. Polysaccharides reduced OTUs diversity and significantly (p < .01) remodeled GM compositions. Bacteria that have been reported for anti-obesity, SCFA-producer, polysaccharides/xylan degradation, and lactic acid production were significantly (p < .05) abundant in mice treated with polysaccharides. We conclude that these polysaccharides act as prebiotics to modulate GM composition thereby potentially contribute to the health promotion effects of G. lucidum and P. cocos.