Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by disrupted epidermal barrier function and excessive immune activation. Conventional treatments using corticosteroids and immunosuppressants provide only temporary relief and often induce adverse side effects, highlighting the need for novel, effective therapy. In this study, we investigated the therapeutic potential of neural stem cell-derived extracellular vesicles (NSC-EVs) derived from NSC-derived conditioned medium (NSC-CM) in modulating inflammatory responses in AD. In vitro experiments using human keratinocytes (HaCaT) and murine macrophages (RAW264.7) showed that both NSC-CM and NSC-EVs can significantly decrease the expression of proinflammatory cytokines and chemokines, inhibit the phosphorylation of NF-κB, and reduce inducible nitric oxide synthase. In addition, topical application of NSC-CM and NSC-EVs alleviated atopic symptoms, reduced mast cell infiltration, and improved skin barrier integrity in a 2,4-dinitrochlorobenzene-induced AD mouse model. Proteomic analysis identified key proteins related to immune regulation and extracellular matrix remodeling in NSC-EVs, indicating their active role in mitigating inflammation and tissue repair. Altogether, our results demonstrated the potent anti-inflammatory effects of NSC-EVs, highlighting their potential to be a novel and effective therapeutic option for AD and other inflammation-related diseases.