Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease, and the pathogenesis is not completely understood. Although there are some mouse models of AD, it is not easy to establish model to represent the natural AD development in human. In this study, we developed an AD model based on outside-inside theory and investigated the effect of Lactobacillus rhamnosus (Lcr35), which have known as an immune modulator in allergic diseases. SKH-1 hairless mice underwent three 1-week exposures (separated by 2-week intervals) to an ovalbumin (OVA) or saline (control) patch at the same site to develop the mouse model of AD. Lcr35 (1 × 10(9) CFU) was administered orally every day from 1 week before the first sensitization until the end of the study. The AD model induced erythematous and itchy skin, increasing TEWL and increasing skin inflammation as assessed by histology in the mice. Oral Lcr35 attenuated all disease parameters previously mentioned. OVA-specific IgE and skin expression of interleukin-4 (IL-4) and thymic stromal lymphopoietin (TSLP) increased in AD mice, but were reduced in AD mice treated with Lcr35. Moreover, Lcr35 treatment led to an increase in CD4(+) CD25(+) Foxp3(+) Treg cells in the mesenteric lymph nodes of AD mice. In conclusions, based on the 'outside-inside' theory, topical allergen may induce AD without skin injury. Oral application of Lcr35 prevented the development of AD in this model by suppressing production of the inflammatory cytokines, IL-4 and TSLP in the skin via a mechanism that may involve CD4(+) CD25(+) Foxp3(+) Treg cells.