Atopic dermatitis (AD) is a chronic skin disease with severe inflammation and pruritus. Traditional Kampo medicine Orengedokuto improves AD symptom in human patients and atopy-like symptoms in AD mouse model. Berberine is a major component of Orengedokuto. This study investigated the effects and molecular mechanisms of berberine on AD-like symptoms in mice. In NC/Nga mice with atopy-like dermatitis (dermatitis mice), intermittent oral administrations of berberine inhibited skin symptom, itching, cutaneous infiltration of eosinophils and mast cells, and the cutaneous expression of eotaxin, macrophage migration inhibitory factor (MIF) and IL-4. Berberine also inhibited both IL-4/MIF-induced eotaxin in fibroblasts and allergen-induced MIF and IL-4 in mast cells. In mast cells, the GeneChip^® microarray analysis showed that antigen increased the expression of EIF3F and MALT1, inhibited by berberine. The regulation of these factors by siRNAs for them showed antigen-induced the expression of MIF and IL-4. These results suggest that berberine inhibits AD-like symptoms through at least downregulation of EIF3F and MALT1 in mast cells.