AIM: To investigate the effect and action mechanism of total flavonoids isolated from the leaves of MATERIALS AND METHODS: The 3-month-old female rats were randomly divided into six groups: sham operation group, OVX model group, estradiol group, TFEL low (TFEL-L) (50), mid (-M) (100) and high (-H) (200 mg/kg/d) dose groups. After 13 weeks of treatment, the rats were sacrificed to measure bone turnover markers, related tissue biochemical indices, microstructure parameters, and osteoclastogenesis promotor RANKL and inhibitor OPG expression levels. Additionally, fecal samples were obtained for high-throughput sequencing to analyze the intestinal flora. RESULTS: Oral administration of TFEL for 13 weeks increased the serum level of bone formation marker PINP and decreased the level of bone resorption marker NTX-I. The femoral microstructure parameters of the TFEL-M and TFEL-H groups were significantly improved compared with the OVX group, which were also confirmed by H&E histological staining. High-throughput sequencing indicated that TFEL may regulate the composition of intestinal flora and intestinal microecology. CONCLUSION: TFEL can prevent osteoporosis in OVX rats and has no toxic side effects. Meanwhile, TFEL can increase the diversity and improve the composition of intestinal flora in OVX rats.