Extracellular vesicles (EVs) play an important role in biological functions and may feature innate therapeutic potential for diseases. In the present study, EVs released by osteoblasts at different stages of the mineralization process were investigated for their potential ability to promote bone formation. Results showed that the characteristics of EVs of mineralizing osteoblasts changed with regularity. EVs derived from the mid-to-late differentiation stage remarkably promoted osteoblast differentiation of bone marrow-derived mesenchymal stem cells and improved osteoporosis in ovariectomized mice. The findings also revealed that the effect of EVs on osteogenesis was related with the maturity of matrix vesicles (MVs), a kind of EVs selectively released by mineralizing-related cells. Nevertheless, only the EVs from the mid-to-late stage showed osteoinductive properties, Synthetic cartilage lymph (SCL) treatment of EVs from the middle stage could promote MV maturation but showed no effect on osteoinduction. Additionally, EVs derived at the middle and mid-to-late stages showed innate bone-targeting potential. Collectively, this study demonstrated that EVs released by osteoblasts at the mid-to-late differentiation stage markedly enhance osteogenesis. Our findings present the prospective use of osteoblast-released EVs in bone tissue engineering. •The characteristics of extracellular vesicles of osteoblasts change with regularity.•Extracellular vesicles at mid-to-late differentiation stage enhance osteogenesis.•Mid-to-late-stage vesicles own osteoinductive and bone-targeting properties.•Matrix vesicles make up a certain proportion of extracellular vesicles.•Maturity of matrix vesicles is related to pro-osteogenesis，but not osteoinduction.