Atopic dermatitis (AD) is an inflammatory skin disease in which type 2 allergic inflammation plays a critical role. In this study, the anti-inflammatory effect of conditioned media from human umbilical cord blood-derived mesenchymal stem cells (USC-CM) was investigated in order to apply it as an effective treatment with a low risk of side effects that can overcome the limitations of AD treatment which is currently in use. We found that USC-CM has various growth factors and cytokines associated with anti-inflammatory effect. RT-PCR and ELISA analysis showed that USC-CM inhibited the levels of type 2 cytokine and chemokine Thymus and activation-regulated chemokine (TARC), TNF-α and IL-6 in TNF-α/IFN-γ-stimulated HaCaT cells. In addition, USC-CM inhibited IL-4 and IL-13 levels in Th2 cells. Therefore, the results of our study demonstrated that USC-CM has anti-inflammatory effect in TNF-α/IFN-γ-stimulated HaCaT cells which associated with the inhibition of the immunoglobulin (IgE) secretion by activating B cell line. Our In vivo results showed that when the USC-CM was applied to lesions of patients with the mild AD for 4 weeks, the skin barrier was strengthened by increasing the level of Corneometer and decreasing the value of transepidermal water loss (TEWL). In conclusion, the results suggest that USC-CM may have therapeutic effect for AD as cosmetics and drug materials.