Atopic dermatitis (AD) is a common inflammatory skin disease characterized by a complex, heterogeneous pathogenesis including skin barrier dysfunction, immunology, and pruritus. Although epidermal growth factor (EGF) is essential for epithelial homeostasis and wound healing, the effect of EGF on AD remains to be explored. To develop a new therapy for AD, the anti-AD potential of EGF was investigated by inducing AD-like skin lesions in NC/Nga mice using 2,4-dinitrochlorobenzene (DNCB). EGF was administrated to NC/Nga mice to evaluate its therapeutic effect on DNCB-induced AD. EGF treatment improved dermatitis score, ear thickness, epidermal hyperplasia, serum total immunoglobulin E level, and transepidermal water loss in NC/Nga mice with DNCB-induced AD. In addition, levels of skin barrier-related proteins such as filaggrin, involucrin, loricrin, occludin, and zonula occludens-1 (ZO-1) were increased by EGF treatment. These beneficial effects of EGF on AD may be mediated by EGF regulation of Th1/Th2-mediated cytokines, mast cell hyperplasia, and protease activated receptor-2 (PAR-2) and thymic stromal lymphopoietin (TSLP), which are triggers of AD. Taken together, our findings suggest that EGF may potentially protect against AD lesional skin via regulation of skin barrier function and immune response.