BACKGROUND Recently, we found that Staphylococcus aureus produces extracellular vesicles (EV) that contain pathogenic proteins. Although S. aureus infection has been linked with atopic dermatitis (AD), the identities of the causative agents from S. aureus are controversial. We evaluated whether S. aureus-derived EV are causally related to the pathogenesis of AD. METHODS Extracellular vesicles were isolated by the ultracentrifugation of S. aureus culture media. The EV were applied three times per week to tape-stripped mouse skin. Inflammation and immune dysfunction were evaluated 48 h after the final application in hairless mice. Extracellular vesicles-specific IgE levels were measured by ELISA in AD patients and healthy subjects. RESULTS The in vitro application of S. aureus EV increased the production of pro-inflammatory mediators (IL-6, thymic stromal lymphopoietin, macrophage inflammatory protein-1α, and eotaxin) by dermal fibroblasts. The in vivo application of S. aureus EV after tape stripping caused epidermal thickening with infiltration of the dermis by mast cells and eosinophils in mice. These changes were associated with the enhanced cutaneous production of IL-4, IL-5, IFN-γ, and IL-17. Interestingly, the serum levels of S. aureus EV-specific IgE were significantly increased in AD patients relative to healthy subjects. CONCLUSIONS These results indicate that S. aureus EV induce AD-like inflammation in the skin and that S. aureus-derived EV are a novel diagnostic and therapeutic target for the control of AD.