BACKGROUND: β-Sitosterol, a prominent phytosterol present in numerous plant species, has been extensively studied for its potential health benefits, such as lipid-lowering, anxiolytic, and anti-inflammatory properties. Recently, the benefit of β-sitosterol on bone metabolism has been noted. The objective of the current study was to examine the impact of β-sitosterol on the skeletal system. METHODS: Network pharmacology and molecular docking were used to predict how β-sitosterol may be used to treat osteoporosis. Cytotoxicity tests were conducted with different concentrations of β-sitosterol. The ability of β-sitosterol to inhibit osteoclast formation and function was evaluated, along with its potential molecular mechanism. An ovariectomized mouse model was used to assess the preventive effect of β-sitosterol on bone loss. RESULTS: Network pharmacology analysis suggested that β-sitosterol could be a potential therapeutic treatment for osteoporosis by regulating the cAMP signaling pathway. β-sitosterol dose-dependently inhibited osteoclast differentiation and function without obvious cytotoxicity. Specifically, 20 μM β-sitosterol could obviously repress the number and size of osteoclasts, decrease the formation of F-actin belts, and reduce the bone-resorbing activity of osteoclasts. Some key signaling mediators, including PKA, c-Jun, NFATc1, p-CREB, and NF-κB, were downregulated by β-sitosterol. β-sitosterol acted by attenuating the cAMP and NF-κB signaling pathways. In vivo experiments confirmed β-sitosterol protected ovariectomy-induced bone loss though suppressing osteoclastic bone resorption. CONCLUSION: β-sitosterol could inhibit the production and function of osteoclasts in vitro and reverse ovariectomy-induced bone loss. Thus, β-sitosterol could be a potential supplement for diseases with active bone resorption such as osteoporosis.