Of the biogenic polyamines, spermidine is a natural constituent of living cells and organisms. Spermidine is associated with regulation of cell growth, proliferation and differentiation, and with the suppression of oxidation and inflammation. Atopic dermatitis (AD) is a chronic inflammatory skin disease that has a complex and multiple pathogenesis, which includes genetic abnormality, modified or abnormal immune response and the production of nitric oxide and reactive oxygen species. We investigated whether spermidine can relieve AD-like clinical manifestation induced by the continual application of 2,4-dinitrofluorobenzene (DNFB) in NC/Nga mice. Spermidine at concentrations of 1 or 10 mg/kg reduced increasing ear swelling and attenuated oedema, haemorrhage and hyperkeratosis in AD-like skin lesions. Repetitive application of DNFB induced inflammatory cell infiltration to skin lesions, whereas intraperitoneal injection of spermidine inhibited DNFB-evoked infiltration of eosinophils, mast cells and T lymphocytes. Furthermore, spermidine suppressed mast cell degranulation and production of interferon-gamma by activated CD4+ T cells in AD-like skin lesions. Spermidine may be a potential therapeutic agent for treatment of AD.