Background : Selaginella tamariscina has been traditionally used in Korea for treating hematochezia, hematuria, and prolapse of the anus. The aim of this study was to evaluate the inhibitory effect of Selaginella tamariscina water extract (ST-WE) on osteoclast differentiation, and to determine the underlying molecular mechanism. Materials and Methods : RAW264.7 cells were used as a model to examine receptor activator for the nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. Expression of osteoclastic genes and transcription factors was evaluated by real-time quantitative polymerase chain reaction (QPCR). Activation of the mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and NF-κB were determined by Western blot analysis. Results : ST-WE significantly inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and formation of multinucleated osteoclasts in RAW264.7 cells. ST-WE also significantly inhibited the RANKL-induced mRNA expression of TRAP, cathepsin K, and the d2 isoform of vacuolar ATPase V(0) domain (ATPv0d2) gene. In addition, ST-WE inhibited the RANKL-induced phosphorylation of ERK, JNK, and p38, phosphorylation of I-κBα and NF-κB p65, and the expression of transcription factors c-fos, Fra-2, and nuclear factor of activated T cells 1. Furthermore, ST inhibited the bone resorptive activity of osteoclasts. Conclusion : ST-WE might have beneficial effects on bonedisease by inhibiting osteoclastogenesis and osteoclastic activity.