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• Hispolon is a bioactive fungal compound isolated form Phellinus , widely used in oriental medicine for various clinical complications. • Hispolon significantly inhibited the RANKL-induced osteoclastogenesis without any cytotoxicity. • Hispolon inhibited the RANKL-induced osteoclastogenesis through inhibition of NF-KB and MAPK signaling and subsequent inhibition of c-FOS and NFATc1. Hispolon (HISP) is a bioactive compound isolated from Phellinu linteus. It has various pharmacological activities, including antioxidant, anti-inflammatory, and anti-cancer. However, its anti-osteoclastogenic activity has not yet been reported. Hence, in the current study, we have explored the anti-osteoclastogenic activity of HISP and elucidated the molecular mechanisms. HISP inhibited the RANKL induced differentiation of RAW 264.7 cells into osteoclasts in a dose-dependent manner. Mechanistic studies showed that HISP inhibited RANKL-mediated activation of NF-κB and MAPK signaling pathways in osteoclast precursors RAW 264.7 cells. In addition, Hispolon also downregulated the expression of master transcriptional factors essential for osteoclast differentiation, such as NFATc1 and c-FOS. In conclusion, these findings establish molecular mechanisms behind the anti-osteoclastogenic activity of HISP. [ABSTRACT FROM AUTHOR]