Osteoporosis, characterized by excessive osteoclast activation, is mediated through the RANKL/RANK/OPG signaling axis. While flavonoids from Eucommia ulmoides (EU) have demonstrated anti-osteoclastogenic activity, their atomic-level mechanisms remain elusive. Here, we investigated six EU-derived flavonoids (cyrtominetin, quercetin, syringetin, genistein, ombuin, and kaempferol) targeting RANKL using integrated computational approaches. Molecular docking revealed strong binding affinities (Total_Score >
4.0) for all compounds, with cyrtominetin exhibiting the highest affinity (-50.205 kJ/mol via MM-PBSA), primarily through hydrogen bonds with Gly178, His180, Lys181, and Asn295. Moreover, most flavonoids interacted with RANKL by forming strong hydrogen bonds with Gly178 and Asn295, exhibiting higher binding affinity that was identified as essential for the activity. All-atom molecular dynamics simulations (100 ns) confirmed complex stability, demonstrating: low RMSD fluctuations (<
4.0 Å) and compact Rg values (16.0-17.0 Å). Notably, binding free energy decomposition identified both electrostatic and van der Waals contributions as critical for stabilization. These results identify cyrtominetin as a promising lead compound for RANKL inhibition, providing structural insights for designing flavonoid-based therapeutics against osteoporosis.