Atopic dermatitis, a chronic inflammatory skin disease characterized by relapsing eczema and intense prurigo, requires effective and safe pharmacological therapy. In this study, we examined the efficacy of ointment containing NF-kB decoy oligodeoxynucleotides (ODN) on atopic dermatitis lesions in NC/Nga mice, which are characterized by the spontaneous onset of atopic dermatitis in conventional conditions. Topical administration of NF-kB decoy ODN twice a month resulted in a significant reduction in clinical skin condition score and marked improvement of histological findings. Reduction of the atopic skin condition by NF-kB decoy ODN was accompanied by a significant decrease in migration of mast cells into the dermis and an increase in apoptotic cells. Here, we demonstrated the successful treatment of atopic dermatitis with ointment containing NF-kB decoy ODN in a mouse model, promising new therapy for atopic dermatitis.