Anti-allergic effects of galactooligosaccharide (GOS), which is found in breast milk and frequently added to food for promoting health, were evaluated in a human-like mouse model of atopic dermatitis (AD). NC/Nga mice were fed 5.5% GOS for 8 weeks, and we examined whether this treatment suppressed the development of AD-like skin lesions in these mice. Mice fed GOS exhibited significantly less symptoms of dermatitis, reduced scratching frequency, and lower levels of serum total immunoglobulin E compared to control. At the end of the 8-week-experimental period, spleens were removed, and the splenocytes were stimulated with phorbol 12-myristate 13-acetate and ionomycin, following which production of cytokines and a chemokine was analyzed. Elevated levels of Th1 cytokines such as interferon-gamma were observed in splenocytes from GOS-fed mice. However, the levels of Th2 cytokines such as interleukin (IL)-13 were unchanged. Furthermore, GOS inhibited the production of inflammatory cytokines such as IL-1beta, IL-6, IL-17, and tumor necrosis factor-alpha but enhanced production of immunomodulatory IL-10. The results indicate that GOS effectively blocked AD-like skin lesions in the mice by at least partly inducing production of IL-10 and suppressing the production of cytokines such as IL-17, which are involved in skin inflammation.