Background and Purpose: 2-Methoxystypandrone (2-MS) is a naphthoquinone isolated from Polygonum cuspidatum, a Chinese herb used to treat bone diseases. Here we have determined whether 2-MS antagonised osteoclast development and bone resorption. Experimental approach: RAW264.7 cells were treated with receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) to induce differentiation into osteoclasts. RT-PCR and Western blot were used to analyse osteoclast-associated gene expression and signalling pathways. KEY RESULTS The number of multinuclear osteoclasts, actin rings and resorption pit formation were markedly inhibited by 2-MS, targeting osteoclast differentiation at an early stage and without significant cytotoxicity. The anti-resorption effect of 2-MS was accompanied by decreasing dendritic cell-specific transmembrane protein and matrix metalloproteinase-9 (MMP-9) mRNA expression. RANKL-increased MMP-9 gelatinolytic activity was also attenuated by concurrent, but not by subsequent addition of 2-MS. 2-MS markedly inhibited not only the RANKL-triggered nuclear translocations of NF-κB, c-Fos and nuclear factor of activated T cells c1 (NFATc1), but also the subsequent NFATc1 induction. Degradation of IκB and phosphorylation of mitogen-activated protein kinases were also suppressed. RANKL facilitated the formation of singaling complexes of tumour necrosis factor receptor-associated factor 6 and transforming growth factor β-activated kinase 1 (TRAF6-TAK1), important for osteoclastogenesis and formation of such signalling complexes was prevented by 2-MS. Conclusions and implications: The anti-osteoclastogenic effects of 2-MS could reflect the block of RANKL-induced association of TRAF6-TAK1 complexes with consequent decrease of IκB-mediated NF-κB and mitogen-activated protein kinases-mediated c-Fos activation pathways and suppression of NFATc1 and other gene expression, essential for bone resorption.