This study investigated the effect of Cornus officinalis vinegar on gut microbiota and hepatic lipid droplet in nonalcoholic fatty liver disease (NAFLD) model mice. The C57BL/6J male mice were divided into three treatment groups: Control group (CON group) fed with a purified diet; high fat diet (HFD) group and C. officinalis vinegar treatment (CVT) group fed with high fat diet. After 10 weeks, the CVT group was orally administered with C. officinalis vinegar once a day for 6 weeks. We found that C.officinalis vinegar improved gut microbiota, significantly reduced body weight and liver injury of HFD-fed mice. Observation of histopathology and ultrastructure showed that C.officinalis vinegar improved histological changes, reduced size and number of lipid droplets. Additionally, C.officinalis vinegar significantly inhibited the expression of perilipin 2 (PLIN-2) and increased the expression of adipose triglyceride lipase (ATGL). In conclusion, C. officinalis vinegar could exert beneficial effects on NAFLD through modulation of gut microbiota and lipid droplets.