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<b>Objective:</b> This study aimed to explore the regulatory effect of <i>Lactarius hatsudake</i> polysaccharide on intestinal flora. <b>Methods:</b> LHP was extracted from <i>L. hatsudake</i> by hot water. LHP-1 and LHP-2 were further purified and characterized. The effects of LHPs on body weight and intestinal microbiome composition were studied in antibiotic-induced mice. <b>Results:</b> LHP-1 was mainly composed of mannose, glucose and galactose. The molar mass ratio of LHP-1 was 3.44∶61.10∶1.22. LHP-2 is mainly composed of mannose and glucose, with a molar mass ratio of 3.84∶153.00. It has a "neuronal" structure and a chain structure region. LHP intervention can restore the structure and diversity of intestinal flora by increasing beneficial bacteria such as <i>Bacteroides</i> and <i>Akkermansia</i> and reducing harmful bacteria such <i>Enterococcus</i> and <i>Brevundimonas</i>. In particular, the increase of the beneficial bacteria <i>A. muciniphila</i> indicates the great potential of LHP in the regulation of human intestinal flora. <b>Conclusion:</b> Polysaccharide from <i>Lactarius hatsudake</i> can improve intestinal metabolism as potential prebiotics.