Background: As the main metabolites of ginsenosides, 20(S, R)-protopanaxadiol [PPD(S, R)] and 20(S, R)-protopanaxatriol [PPT(S, R)] are the structural basis response to a series of pharmacological effects oftheir parent components. Although the estrogenicity of several ginsenosides has been confirmed,however, the underlying mechanisms of their estrogenic effects are still largely unclear. In this work,PPD(S, R) and PPT(S, R) were assessed for their ability to bind and activate human estrogen receptor a(hERa) by a combination of in vitro and in silico analysis. Methods: The recombinant hERa ligand-binding domain (hERa-LBD) was expressed in E. coli strain. Thedirect binding interactions of ginsenosides with hERa-LBD and their ERa agonistic potency wereinvestigated by fluorescence polarization and reporter gene assays, respectively. Then, molecular dynamicssimulations were carried out to simulate the binding modes between ginsenosides and hERa-LBDto reveal the structural basis for their agonist activities toward receptor. Results: Fluorescence polarization assay revealed that PPD(S, R) and PPT(S, R) could bind to hERa-LBDwith moderate affinities. In the dual luciferase reporter assay using transiently transfected MCF-7 cells,PPD(S, R) and PPT(S, R) acted as agonists of hERa. Molecular docking results showed that these ginsenosidesadopted an agonist conformation in the flexible hydrophobic ligand-binding pocket. The stereostructureof C-20 hydroxyl group and the presence of C-6 hydroxyl group exerted significant influenceon the hydrogen bond network and steric hindrance, respectively. Conclusion: This work may provide insight into the chemical and pharmacological screening of noveltherapeutic agents from ginsenosides.