Scope: Human breast milk-derived extracellular vesicles (HMEVs) are known to support health through various physiological functions such as immune regulation, cell regeneration, and inflammation suppression, as well as having potential in the treatment of diseases, however, research on bone growth is insufficient. Method and results: This study examines the effects of extracellular vesicles derived from donated human breast milk (HMEVs) on osteoblast differentiation and mineralization, aiming to uncover their potential role in osteoporosis prevention and treatment. The findings reveal that HMEVs significantly enhance osteoblast proliferation, differentiation, and mineralization, as evidenced by increased expression of bone formation markers such as osteopontin (OPN), alkaline phosphatase (ALP), and pro-collagen type I (pro-COL I). These effects are mediated via the bone morphogenetic protein 2 (BMP2) and mitogen-activated protein kinase (MAPK) signaling pathways. Conclusion: Consequently, HMEVs show promise as therapeutic agents for preventing osteoporosis by promoting bone cell differentiation and mineralization through BMP2 and MAPK signaling pathways.