Abstract: Background　Staphylococcus aureus, a common pathogen in bone infections, can excessively activate osteoclasts through various pathways, leading to osteolytic destruction. However, whether its secreted outer membrane vesicles play a pivotal pathogenic role in this process remains unclear, and the underlying mechanisms warrant further investigation. Objective　To systematically define the osteoclast-activating effects and phenotypic remodeling induced by Staphylococcus aureus extracellular vesicles, and to explore the dose-dependent association between extracellular vesicle burden and bone destruction, together with the underlying biological mechanisms. Methods　Staphylococcus aureus extracellular vesicles were isolated using a tangential flow filtration system combined with ultracentrifugation, and their morphology, size distribution, and zeta potential were characterized by transmission electron microscopy and nanoparticle tracking analysis. Bone marrow–derived monocytes/macrophages were isolated from mice and induced to differentiate into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Cells were treated with different concentrations of Staphylococcus aureus extracellular vesicles (0, 0.6, 6, and 60 μg/mL) or vehicle control. Osteoclast differentiation and cellular viability were evaluated by tartrate-resistant acid phosphatase (TRAP) staining and CCK-8 assays, respectively, while the expression of osteoclast-related functional genes was assessed by quantitative real-time PCR. Transcriptomic profiling was performed to compare the 6 μg/mL extracellular vesicle-treated group with the control group, followed by pathway enrichment analysis. Furthermore, an in vivo mouse model was established by intramedullary injection of extracellular vesicles into the femur, with phosphate-buffered saline as a control. Bone destruction and osteoclast activation were evaluated using micro-computed tomography and cathepsin K (CTSK) immunofluorescence staining. Results　Staphylococcus aureus extracellular vesicles with typical vesicular morphology were successfully isolated, exhibiting a size distribution predominantly ranging from 100 to 400 nm and a zeta potential between -30 and -10 mV, indicating good colloidal stability. In vitro experiments demonstrated that, compared with the control group, extracellular vesicle treatment significantly promoted osteoclast differentiation and multinucleation, with the number and area of TRAP–positive cells increasing in a concentration-dependent manner and peaking at 6 μg/mL (P＜0.01). CCK-8 assays revealed that extracellular vesicles at concentrations of 0.6 - 6 μg/mL significantly enhanced osteoclast viability (P＜0.01), whereas this stimulatory effect was attenuated at 60 μg/mL. RT-qPCR analysis showed that the mRNA expression levels of Acp5, Dc-stamp, Ctsk, and Mmp9 were significantly upregulated following extracellular vesicle treatment, with the highest expression observed in the 6 μg/mL group (all P ＜0.01). Transcriptomic analysis identified 161 differentially expressed genes, with upregulated genes predominantly enriched in inflammatory responses, Toll-like receptor signaling pathways, and osteoclast differentiation-related pathways. In vivo, extracellular vesicle-treated mice exhibited a significant reduction in femoral bone mineral density (~23%), accompanied by decreases in trabecular number (~30%) and trabecular thickness (~20%) compared with controls (all P＜0.001). Consistently, CTSK-positive signals in bone tissue were markedly increased in the extracellular vesicle-treated group (P＜0.001), indicating markedly elevated osteoclast activity. Conclusion　Staphylococcus aureus extracellular vesicles can establish a localized pro-inflammatory microenvironment by activating the Toll-like receptor pathway, thereby driving the activation and differentiation of osteoclasts and leading to the development of infectious bone destruction.