Annexin A3 (ANXA3), a member of Annexin family, is reported to mediate membrane transport and cancer development. However, the effect of ANXA3 on osteoclast formation and bone metabolism is still unclear. In this study, we found that knockdown of ANXA3 can significantly inhibit receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation through NF-κB signaling. ANXA3 downregulation abrogated the expression of osteoclast-specific genes, including Acp5, Mmp9 and Ctsk in osteoclast precursors. Moreover, lentiviral of shRNA against ANXA3 reversed the bone loss in osteoporosis using ovariectomized mice model. Mechanistically, we found that ANXA3 directly bound to RANK and TRAF6 to accelerate osteoclast differentiation by promoting their transcription and limiting degradation. In conclusion, we propose a fundamentally novel RANK-ANXA3-TRAF6 complex to effectively modulate the formation and differentiation of osteoclast to manipulate bone metabolism. The ANXA3-targeted therapeutic strategy may provide new insight for bone degrading-related diseases prevention and treatment.