Objective: This study aimed to explore the regulatory effect of Lactarius hatsudake polysaccharide on intestinal flora. Methods: LHP was extracted from L. hatsudake 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. Results: 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 Bacteroides and Akkermansia and reducing harmful bacteria such Enterococcus and Brevundimonas. In particular, the increase of the beneficial bacteria A. muciniphila indicates the great potential of LHP in the regulation of human intestinal flora. Conclusion: Polysaccharide from Lactarius hatsudake can improve intestinal metabolism as potential prebiotics.