Background <p>Bone marrow mesenchymal stem cells (BM-MSCs) ameliorate various orthopedic diseases through the secretion of extracellular vesicles (EVs). This study investigates the effects of FTO-overexpressing BM-MSCs-derived EVs (FTO-EVs) on osteocyte autophagy and pyroptosis in <i>Staphylococcus aureus</i>-associated osteomyelitis (OM).</p> Methods <p>The expression levels of N6-methyladenosine regulators were investigated in bone tissues and serum EVs obtained from patients with OM. Osteoblast MC3T3-E1 and osteocyte MLO-Y4 cells were induced with <i>Staphylococcal</i> Protein A (SpA) to simulate in vitro OM models, and murine OM models were established by bone drilling followed by local injection of <i>Staphylococcus aureus</i> suspension, both of which were treated with EVs.</p> Results <p>Fat mass and obesity-associated protein (FTO) was downregulated in the bone tissues or serum EVs from OM patients compare to healthy control. Compared with negative control EVs, FTO-EVs significantly increased FTO expression, elevated cell viability, and inhibited cell death, ATG2A/ATG14-mediated autophagy and NLRP3-mediated pyroptosis in SpA-induced MC3T3-E1 and MLO-Y4 cells. Treatment with FTO-EVs significantly ameliorated bone damage in OM model mice, upregulated the expression of pro-survival proteins in bone tissue, while downregulating key markers of autophagy and pyroptosis. Mechanistically, FTO-EVs N6-methyladenosine dependently downregulated the expression of ATG2A, ATG14 and NLRP3 to modulate autophagy and pyroptosis in SpA-induced MC3T3-E1 and MLO-Y4 cells.</p> Conclusions <p>In summary, this study confirms the role of BM-MSCs-derived FTO-EVs in inhibiting the progression of <i>Staphylococcus aureus</i>-related OM both in vivo and in vitro, providing promising therapeutic strategies for clinical treatment.</p>