Background <p>Periodontitis is a long-lasting inflammatory disorder of structures attached, which is significantly influenced by insufficient or dysregulated immune responses. Mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) are a promising therapeutic modality. This study explores the therapeutic efficacy of EVs derived from miR-146a-overexpressing bone marrow-derived mesenchymal stem cells (BMSCs) in a ligature-induced periodontitis model.</p> Results <p>BMSCs were transfected to overexpress miR-146a, and their EVs were isolated. Periodontitis was induced in 30 female C57Bl/6 mice and divided into three groups: a control group receiving PBS, a miR-control EV-treated group, and a miR-146a-EV-treated group. EVs were administered subgingivally, and clinical parameters were evaluated. Levels of proinflammatory cytokines interleukin (IL)-1β, IL-8, tumor necrosis factor (TNF)-α, and IL-6, as well as anti-inflammatory cytokines IL-10, IL-4, and transforming growth factor (TGF)-β, were quantified using enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (PCR). Phosphorylated p38, JNK, ERK1/2, and NF-κB p65 were also quantified by ELISA, and TRAF6 and IRAK1 mRNA and protein levels were assessed to evaluate inflammatory signaling pathways. In the gingival tissue and systemic circulation, the proinflammatory cytokines IL-1β, IL-8, TNF-α, and IL-6 were significantly downregulated, while the levels of the anti-inflammatory mediators IL-10, IL-4, and TGF-β were significantly increased in the miR-146a-EV group when compared with the control group. miR-146a-EV treatment significantly reduced MAPK and NF-κB activation while downregulating TRAF6 and IRAK1 expression in gingival tissues.</p> Conclusion <p>miR-146a-transduced MSC-derived EVs ameliorated ligature-induced periodontitis by modulating local and systemic cytokine expression. These results indicate that miR-146a-EVs can be a novel, cell-free therapy for the treatment of periodontitis.</p>