Excessive　osteoclast　activity　could　cause　skeletal　diseases　including　osteoporosis.　Additionally,　autophagy　plays　an　initial　role　in　osteoclast　differentiation　and　function.　Ginkgolide　B　(GB),　a　key　compound　in　Ginkgo　biloba,　improves　bone　mass　and　suppresses　mature　osteoclast　formation　in　vitro.　This　study　examines　the　role　of　GB　role　in　regulating　osteoclast　formation　via　autophagy.　Using　murine　bone　marrow-derived　macrophages　in　vitro,　we　explored　GB＇s　effects　on　autophagy　and　osteoclast　formation.　We　also　assessed　bone　loss　prevention　in　an　ovariectomized　(OVX)　mouse　model　using　GB　combined　with　an　autophagy　inhibitor.　Tartrate-resistant　acid　phosphatase　staining　was　used　to　observe　osteoclast　formation.　Autophagy-related　proteins　and　intracellular　microtubule　associated　protein　1　light　chain　3　beta　puncta　were　observed　using　western　blotting　and　immunofluorescence.　The　impact　of　GB　on　OVX　mice　was　evaluated　using　micro-computed　tomography　and　hematoxylin　and　eosin　staining.　GB　directly　promoted　autophagy　in　osteoclast　precursors　(OCPs),　but　inhibited　osteoclast　differentiation　by　reducing　receptor　activator　of　nuclear　factor　kappa　B　ligand　(RANKL)-induced　autophagy.　GB　inhibits　the　phosphorylation　of　RANKL　downstream　signaling　pathways,　such　as　Jun　N-terminal　kinase　(JNK),　p38,　and　extracellular　signal-regulated　kinase　(ERK).　Anisomycin　(ANI),　a　JNK　activator,　reversed　GB＇s　inhibitory　effects　on　RANKL-induced　autophagy　and　osteoclastogenesis.　Inhibiting　autophagy　using　3-Methyladenine　(3-MA)　or　small　interfering　RNA　significantly　suppressed　osteoclast　differentiation　both　in　vitro　and　in　vivo.　Our　findings　suggested　that　GB　inhibits　osteoclast　formation　by　decreasing　JNK　phosphorylation　and　autophagy　under　RANKL　stimulation.　Interestingly,　GB　also　directly　promotes　autophagy　in　OCPs.　Thus,　GB　markedly　reduces　osteoclast　differentiation　and　prevents　bone　loss,　with　its　anti-osteoclastogenesis　effect　being　enhanced　by　3-MA.　Accordingly,　inhibiting　GB-induced　direct　autophagy　could　further　increase　its　therapeutic　effect　on　bone　disease　resulting　from　excessive　osteoclast　activity.