The receptor activator of nuclear factor-kappaB ligand (RANKL) plays a critical role in the differentiation and bone resorptive activity of osteoclasts. Recently, the development of anti-resorptive agents from natural substances has become a subject of interest. Therefore, we evaluated the effects of 222 natural compounds on the RANKL-induced tartrate-resistance acid phosphatase (TRAP; a marker for osteoclast differentiation) activity and multinucleated osteoclast formation in RAW264.7 murine macrophage cells. We found that saurolactam was one of the compounds inhibiting the RANKL-induced osteoclastogenesis; it significantly inhibited the RANKL-induced TRAP activity and formation of multinucleated osteoclasts without any cytotoxicity. Interestingly, saurolactam prevented RANKL-induced activation of MAP kinases and NF-kappaB, and mRNA expression of osteoclast-related genes and transcription factors (c-Fos, Fra-2, and NFATc1). We also observed the inhibitory effect of saurolactam on the differentiation of mouse bone marrow-derived macrophages into osteoclasts. Furthermore, saurolactam inhibited the bone resorptive activity of mature osteoclasts with the induction of apoptotic signaling cascade and the inhibition of survival signaling pathways such as c-Src/PI3K/Akt, Ras/ERK, and JNK/c-Jun. In conclusion, although further studies are needed to determine the precise mechanism and biological efficacy of saurolactam in osteoclast-mediated bone disorders, our results demonstrate that saurolactam potentially inhibits osteoclast differentiation by preventing the activation of MAP kinases and transcription factors that consequently affect the regulation of genes required for osteoclastogenesis, and the bone resorptive activity of mature osteoclasts by inhibiting osteoclast survival-related signaling pathways and triggering the apoptotic signaling cascade.