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Objective: To explore the regulating function of the dietary fiber of Agaricus bisporus and the powder of Agaricus bisporus on the human gut microbiota. Methods: Dietary fiber of Agaricus bisporus were extracted by water extraction and alcohol precipitation for human gut microbial fermentation in vitro. The pH, gas production and short-chain fatty acid contents of metabolites were determined. The V4 region of fecal microorganisms was enriched and sequenced by IlluminaPE250 sequencing platform. Results: Taking fructo-oligosaccharide as the positive control group and without dietary fiber as the blank control group, during the in vitro fermentation process, with the increase of time, the pH value of the Agaricus bisporus dietary fiber group decreased from 6.93 to 4.48, and the pH value of the powder of Agaricus bisporus decreased from 6.93 to 4.86. With the increasing of time, compared with the blank group, the gas production of the Agaricus bisporus dietary fiber group and the powder of Agaricus bisporus group increased by 3.4 and 1.9 mL. After 24 hours of in vitro fermentation, it was found that compared with the positive control group, the richness and diversity of the human gut microbiota in the Agaricus bisporus dietary fiber group and the powder of Agaricus bisporus group were relatively higher. Compared with the blank group, dietary fiber of Agaricus bisporus group was more conducive to the production of acetic acid (34.3 mmol/L) and propionic acid (7.6 mmol/L) by human gut microbiota and promoted the growth of beneficial bacteria of Bifidobacterium. The powder of Agaricus bisporus group was more conducive to producing acetic acid (39.4 mmol/L) and promoting the growth of Enterobacter. Compared with the positive control group, the production of propionic acid in the Agaricus bisporus dietary fiber group and the powder of Agaricus bisporus group was relatively higher. Conclusion: Both the dietary fiber of the Agaricus bisporus and the powder of Agaricus bisporus could be effectively utilized by the human gut microbiota to produce short-chain fatty acids and increase the relative abundance of beneficial bacteria. Compared with blank group, dietary fiber of Agaricus bisporus added in experimental group had the best results, which had an effective prebiotic regulation function on human gut microbiota.