To investigate the effect of osteoblast-derived extracellular vesicles (OB-EVs) on the proliferation and differentiation of osteoclasts, and to explore the possible molecular mechanism of extracellular vesicles involved in the communication between osteoblasts and osteoclasts, and to elucidate the specific mechanism of extracellular vesicles interfering with alveolar bone homeostasis. Primary osteoblasts were isolated from newborn mouse calvarial bone and induced by dexamethasone, β glycerin phosphate and ascorbic acid. Osteogenic feature was tested by alkaline phosphatase (ALP) and alizarin red S staining. Extracellular vesicles were isolated by ultracentrifugation from the supernatant of cell culture. The vesicle morphology was observed by transmission electron microscopy, and the characteristic markers of tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (Alix) and cluster of differentiation 9 (CD9) on the surface of extracellular vesicles were identified by Western blotting. Cell counting kit 8 (CCK-8) assay was used to determine the proliferation effect of OB-EVs on mouse mononuclear macrophage RAW264.7 cells. Furthermore, the expression level of specific markers of osteoclast differentiation in RAW264.7 cells was detected by Western blotting after the combined effect of OB-EVs and nuclear factor kappa B receptor activating factor ligand (RANKL). The number of osteoclasts was observed and compared with OB-EVs-treated mouse bone marrow-derived macrophages (BMMs) by tartrate-resistant acid phosphatase (TRAP) staining, and the effect of OB-EVs on osteoclast differentiation was determined. The extracted OB-EVs showed a double-layer cup-like structure with a diameter of 30-150 nm, and TSG101, Alix and CD9 were positively expressed. RAW264.7 cells were stimulated with OB-EVs, and the results of CCK-8 assay showed that high concentration of OB-EVs (more than 20 μg/mL) inhibited cell proliferation (P<0.05). Western blot analysis showed that the expression of osteoclast differentiation marker proteins such as c-Fos, activated T cell nuclear factor (NFATc1) and c-Jun N-terminal kinase (JNK) in RAW264.7 cells was significantly increased, and the promoting effect was enhanced with the increase of OB-EVs concentration (P<0.05). In addition, the combination of OB-EVs and RANKL on BMM showed that the number of TRAP-positive cells was significantly higher than that of the RANKL induction group alone (P<0.05). High concentration of OB-EVs can inhibit the proliferation of RAW264.7 cells, and OB-EVs can promote the differentiation of osteoclast precursor cells into osteoclasts.