Background: Rosae Multiflorae fructus (RMF), known to have anti-inflammatory and antioxidant properties, has been used as a traditional remedy for inflammatory diseases such as arthritis in Eastern Asia. However, its effect on osteoclasts, which play a crucial role in resorptive inflammatory bone diseases, is yet to be elucidated. Methods: The effect of extract of RMF (RMF-E) on receptor activator of nuclear factor-κB ligand (RANKL)mediated osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRAP) staining, real-time polymerase chain reaction and western blot analysis. In addition, RA-NKL-induced Ca 2+-oscillation was also investigated. Results: RMF-E remarkably inhibited TRAP +-osteoclast and resorptive pit formation in a dose-dependent manner. In addition, the expression of c-Fos and nuclear factor of activated T-cells cytoplasmic, known as pivotal transcription factors for osteoclast formation in vitro and in vivo, and that of the osteoclast differentiation markers such as Acp5, Oscar, CtsK, Atp6v0d2, Tm7sf4, and Nfatc1 were significantly decreased by RMF-E treatment during osteoclastogenesis. The inhibitory effect of RMF-E on RANKL-induced osteoclastogenesis was caused by the suppression of p38 mitogen-activated protein kinase activation, and RANKL-induced Ca 2+-oscillation removal via inactivation of Bruton's tyrosine kinase (BTK), and subsequently phospholipase C-γ2. Conclusions: RMF-E negatively regulates osteoclast differentiation and formation. These findings suggest the possibility of RMF-E as a traditional therapeutic agent against osteoclast-related bone disorders such as osteoporosis, rheumatoid arthritis, and periodontitis.