Dysregulation of osteoclasts or excessive osteoclastogenesis significantly –contributes to the occurrence and development of osteolytic diseases, including osteoporosis, inflammatory bone erosion, and tumor-induced osteolysis. The protein-protein interaction between the receptor activator of nuclear factor (NF)-κB (RANK) and its ligand (RANKL) mediates the differentiation and activation of osteoclasts, making it a key therapeutic target for osteoclastogenesis inhibition. However, very few natural compounds exerting anti-osteoclastogenesis activity by inhibiting the RANKL-RANK interaction have been found. Niloticin is a natural tetracyclic triterpenoid compound with anti-viral, antioxidative, and mosquitocidal activities. However, its role in osteoclastogenesis remains unknown. The present study found that niloticin directly binds to RANK with an equilibrium dissociation constant of 5.8 μM, blocking RANKL-RANK interaction, thereby inhibiting RANKL-induced AKT, MAPK (p38, JNK, and ERK1/2), and NF-κB (IKKα/β, IκBα, and p65) pathways activation, and reducing the expression of key osteoclast differentiation-related regulatory factors (NFATc1, c-Fos, TRAP, c-Src, β3-Integrin, and cathepsin K) in osteoclast precursors, ultimately negatively regulating osteoclastogenesis. These findings suggest that niloticin could serve as a novel osteoclastogenesis inhibitor and might have beneficial effects on bone health.