Non-thermal plasma (NTP) has many functional activities such as, sterilization, wound healing and anti-cancer activity. Thus, its application in bio-medical field is expanding quickly. However, the effect of NTP on immune cells and allergic response has not been well studied. In this study, we determined whether NTP could suppress mast cell activation, which is important for allergic response, and ameliorate an atopic dermatitis (AD)-like skin inflammatory disease in mice. Treatment of NTP-treated medium during mast cell activation inhibited IL-6, TNF-α expression and production and suppressed NF-κB activation. We also investigated whether NTP treatment could ameliorate house dust mite (HDM)-induced AD-like skin inflammation in mice. NTP treatment inhibited increases in epithermal thickness and recruitment of mast cells and eosinophils, which are important cell types for the pathogenesis of AD. In addition, Th2 cell differentiation was induced by application of HDM and the differentiation was also inhibited in the draining lymph node of NTP-treated mice. Finally, the expression of AD-related cytokines and chemokines also decreased in NTP-treated mice. Taken together, these results suggest that NTP might be useful in the treatment of allergic skin diseases, such as AD.