Rosa davurica Pall. (RDP) fruits are popularly consumed as beverages and healthy food in China because of their various beneficial activities. In particular, flavonoids are one of the major active ingredients of RDP fruits with predominant pharmacological effects. However, the anti-obesity activities of flavonoids from RDP fruits and their regulation effect on the gut microbiota have not been determined. In the present study, the flavonoid-rich extracts (RDPF) were isolated from RDP fruits and their anti-obesity effects were investigated using a high-fat diet (HFD)-induced obese mouse model. The results showed that RDPF intervention significantly inhibited the body weight, liver weight, kidney weight and epididymal adipose tissue weight of HFD-fed mice without affecting the calorie intake. Plasma lipid levels were also significantly lowered by RDPF treatment. Histological examination showed that RDPF supplementation partially recovered HFD-induced hepatic steatosis in the liver. RDPF also prevented oxidative injury of the liver, as evidenced by the altered superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) levels. The expression levels of CCAAT/enhancer binding protein α (C/EBPα), sterol regulatory element binding protein-1C (SREBP-1C), fatty acid synthase (FAS), acyl-coenzyme A oxidase 1 (ACOX1), peroxisome proliferator-activated receptor (PPARα) and CAT mRNA in the livers of mice were also regulated by RDPF administration. 16S rRNA gene sequence data further indicated that RDPF addition increased the microbial diversity and reshaped the community composition. Intriguingly, RDPF intervention did not exhibit inhibitory tendency toward the ratio of Firmicutes to Bacteroidetes, but markedly decreased the relative abundance of Erysipelotrichaceae. This study provided novel insights into the application of RDPF in the food industry.