Atopic dermatitis (AD) is chronic inflammatory skin disease with sever pruritus. However, the mechanisms of itching are not fully understood. In this study, we demonstrated that the involvement in the receptor subtypes (5-HT receptors) on spontaneous scratching, an itch-related behavior, in mice with atopy-like dermatitis. Male NC/Nga mice with AD-like dermatitis (dermatitis NC mice), which were bred under conventional condition with mites, and male healthy mice (healthy NC mice), which were bred under specific pathogen free condition, were used. An intradermal injection of serotonin, but not tryptophan and histamine, elicited scratching in healthy NC mice. Spontaneous scratching in dermatitis NC mice was inhibited by methysergide (5-HT₂ receptor antagonist), but not cyproheptadine (5-HT_2A receptor antagonist) or ondansetron (5-HT₃ receptor antagonist). An intradermal injection of a-methyl-5-HT (5-HT₂ receptor agonist) or BW723 C86 (5-HT_2B receptor agonist), but not KM-212 (5-HT_2C receptor agonist), induced scratching in healthy NC mice. Taken together, these findings suggest that 5-HT_2B receptor may be involved in the induction of spontaneous scratching in dermatitis NC mice.