Atopic dermatitis (AD) is a pruritic inflammatory skin disease caused by skin barrier dysfunction and immune abnormalities. We have previously reported an AD mouse model using outbred HR-1 hairless mice. However, this mouse strain might be inapplicable to studies requiring genetically modified mice. Therefore, we examined whether AD symptom is reproduced using C57BL/6 (B6) mice, a strain widely used for gene-targeting. Four-week-old female B6 mice were fed a special diet (SD) deficient in unsaturated fatty acids and starch to induce skin barrier dysfunction systemically. Then, house dust mite (HDM) extract was repeatedly applied to the face and back skin of SD-fed mice (SD + HDM mice). SD + HDM mice showed AD-like dry and eczematous lesions and itch-related behavior. In the skin of SD + HDM mice, the numbers of eosinophils and mast cells were increased compared with normal B6 mice. Furthermore, the expression levels of S100a8, Krt16, Il1b, Il13, Il17a, Il19, Cxcl1, and Ccl17 were increased, whereas that of Lor was decreased. The skin gene expression profile in SD + HDM mice more closely resembles that of human AD than well-known other AD mouse models (spontaneous model, NC/Nga mice, or sensitizer (ovalbumin or oxazolone)-induced models). In conclusion, our novel AD model established using B6 mice could be useful for studying AD.