Disruption of the balance between osteoclasts and osteoblasts could lead to bone diseases including osteoporosis. It's well known that RANKL-RANK signaling plays a vital role in activating osteoclasts. Herein, we explored the therapeutic effects of toosendanin (TSN) in osteoporosis, showing that TSN attenuated RANKL-stimulated osteoclastogenesis and osteoclast-specific gene expression in vitro. Bioinformatics predicted that TSN could interfere p38 subunits and regulate the MAPK cascade, and we further verified and demonstrated that TSN significantly inhibited RANKL-induced p38 signaling through western blot. In ovariectomized mouse model, TSN effectively inhibited the formation of TRAP-positive osteoclasts and exhibited protective effect against bone loss. Altogether, these data indicate that TSN targeted p38 activation to inhibit osteoclastogenesis, suggesting the possible therapeutic use of TSN in osteoporosis in the future.