Excessive absorption of osteoclasts will break the balance between osteoclasts and osteoblasts, leading to bone loss, decreased bone density, and increased bone fragility. We have shown that Loureirin B (LrB) can inhibit osteoclasts. In this study, we demonstrated the targeting-inhibitory mechanism of LrB acting on osteoclast precursor. Using SPR, HPLC and MALDI-TOF-MS to capture and analyze the target protein of Loureirin B in bone marrow macrophages (BMMs), we used this method to detect all target proteins that LrB acts on BMMs, and analyzed the distribution and enrichment rate of the target protein by DAVID enrichment analysis. Ledock molecular docking was used to detect the binding of LrB. We used Western Blot for verification. The target proteins of LrB acting on BMMs were Serpine1, Atp6ap1, Dvl1, Rhd, Fzd2, MAPK1, MAP2K2, MAPK3 and so on. MAPK1, MAP2K2 and MAPK3 were the most relevant. LrB treatment attenuated the expression of phosphorylated JNK and p38 kinases of the MAPK signaling pathway. Our research further confirmed that LrB affects the MAPK signaling pathway in BMMs, thereby inhibiting the differentiation of BMMs into osteoclasts. This discovery can confirm the mechanism by which LrB acts on BMMs.© 2022. The Author(s).