Background <p>Atopic dermatitis (AD) is a chronic inflammatory skin disease primarily treated with corticosteroids and dupilumab, a monoclonal antibody targeting interleukin (IL)-4 and IL-13. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are promising alternatives owing to their anti-inflammatory properties. This study compared the therapeutic effects of dupilumab and MSC-EVs in a murine model of AD. We employed clinical, serological, and histological analyses to assess the efficacy of these treatments and investigated their mechanisms in vitro and in vivo.</p> Methods <p>We generated Wharton’s jelly MSC-EVs using a 3D microwell-based platform and evaluated their effects in an AD mouse model. AD was induced by repeated application of 2,4-dinitrochlorobenzene (DNCB) and sodium dodecyl sulfate (SDS). The mice were randomly divided into four groups: healthy (normal), placebo (DNCB + SDS), EV (6 × 10⁸ particles once), and dupilumab (25&#xa0;mg/kg biweekly) groups. Dupilumab and EVs were injected subcutaneously into the dorsal skin of the mice. Dermatitis scores, serum inflammatory markers, and histological analyses were performed to evaluate disease severity and changes at the tissue level. Additionally, tumor necrosis factor (TNF)-α-induced HaCaT cells were utilized for in vitro experiments to investigate the molecular mechanisms of MSC-EV therapy.</p> Results <p>EVs and dupilumab improved the clinical dermatitis score, reduced epidermal thickness, and promoted restoration of the skin barrier in mice with AD. Treatments decreased T helper (Th)2 and pro-inflammatory cytokines, but EVs, unlike dupilumab, effectively suppressed Th1 and Th22 cytokines. EVs suppress Th1 activation through the AKT/NF-κB pathway via microRNA-146a.</p> Conclusions <p>MSC-EVs offer a novel cell-free therapy for AD, demonstrating comparable or superior efficacy to dupilumab, with broader immunomodulatory effects and the advantage of a single-dose administration.</p>