Background: Atopic dermatitis (AD) is an inflammatory skin disorder with severe pruritus. Despite advancements in medicine, therapeutic treatments for AD are still limited. Eupatilin (5,7-dihydroxy-30,40,6-trimethoxyflavone) is one of the lipophilic flavonoids from Artemisia umbelliformis Lam. and Artemisia genipiWeber. Objective: Although it has been reported to act a role in improving inflammation, its action on AD is uncertain. In this study, we examined the role of eupatilin on AD-like skin lesions in NC/Nga mice. Methods: 2,4-dinitrochlorobenzene was repeatedly applied to the ear of NC/Nga mice to produce AD-like skin lesions. Eupatilin (1%, once a day for 5 consecutive days/week) was applied topically for four weeks for the evaluation of its therapeutic effects. Results: 1% eupatilin cream significantly reduced the clinical severity score of AD-like lesions, compared to the vehicle (p<0.005). A histopathological analysis revealed that % eupatilin cream significantly decreased the mast cell infiltration as well as inflammatory cell infiltration, compared to the vehicle (p<0.005). We showed that 1% eupatilin cream significantly reduced the expression of thymic stromal lymphopoietin, tumor necrosis factor-alpha, interleukin-4, and interleukin-19, but not interferon-gamma, compared to the vehicle (p<0.005). Conclusion: Considering the therapeutic reaction of eupatilin on AD-like lesions as in this study, the substance has a promising to be an adjuvant topical agent for the control of AD.