<p>Osteosarcoma (OS) is a leading malignant bone tumor in children and adolescents. Resetting M2-polarized tumor-associated macrophages (TAMs) has emerged as a promising therapeutic target for OS. Brucea javanica has long been used in China for cancer treatment, but its effects on TAMs in OS remain underexplored. Human THP-1 and murine RAW264.7 macrophages were polarized to an M2 phenotype by IL-4 and/or IL-13. The effects of Brucea javanica oil (BJO) and acetone extract (DBA) on TAM repolarization were assessed by qRT-PCR, ELISA, and flow cytometry. BJO’s impact on M2-promoted OS cell proliferation, migration, invasion, and vasculogenic mimicry was evaluated in vitro. BJO were characterized by GC-TOF-MS analysis. Network pharmacology, western blot, and rescue experiments were carried out to uncover the mechanism through which BJO reprogram TAMs. An OS mouse model generated by co-implantation of K7M2 and M2-polarized RAW264.7 macrophages was used to evaluate the effects of BJO on OS growth and TAM reprogramming in vivo. BJO exhibits a stronger capacity than DBA to switch TAMs from M2 to M1 phenotype in a dose-dependent manner, thereby inhibiting M2 macrophage-promoted aggressive behaviors of OS cells in vitro. Nine constituents were identified in BJO by GC-TOF-MS. Mechanistically, BJO inhibits PI3K/AKT pathway in TAMs, thereby relieving its negative regulation of ERK1/2 and NF-κB signaling. In vivo, BJO inhibits OS growth and drives TAMs into an M1 phenotype, while demonstrating minimal systemic toxicity. These findings reveal a novel anti-OS mechanism of BJO, highlighting its potential as an M2-TAM-targeted immunotherapeutic agent.</p> Graphical abstract <p></p>