Objective: The influence of ethanol extracts obtained from Auricularia heimuer fruit body (AHA) on the liver function and the diversity and structure of gut microbiota in high-fat diet mice were explored. Methods: The obese mice model was established by high-fat diet. After feeding for 9 weeks， the changes of physiological and biochemical indicators of mice such as body weight and body length were compared before and after AHA intervention， and the size and number of liver cells were observed by Hematoxylin-eosin (HE) staining. 16S rRNA gene sequencing was used to profile the diversity and structure of intestinal flora in the cecum samples. Results: AHA treatment group significantly decreased the body weight， epididymis fat and subcutaneous fat index of mice (P<0.05)， significantly decreased Lee's index， serum GLU and liver index (P<0.01) and increased the content of HDL-C in the liver (P<0.05). In AHA group， the adipocytes and lipid droplets in mice were observed to become smaller and the nucleus gradually became clear by HE staining. In addition， High throughput sequencing showed that AHA could regulate the diversity and richness of intestinal flora. Conclusion: AHA could significantly prevent nutritional obesity， inhibit fatty liver formation， improve liver tissue， reduce the degree of lesion and regulate the disorder of intestinal flora.