In this study, we evaluated the effect of oral administration of galacto-oligosaccharide (GOS), famous biological molecules that are comprised of galactose and lactose, on ovalbumin (OVA)-induced allergic dermatitis. OVA-induced mice were divided into the OVA-administered group (OVA-C), promethazine hydrochloride-administered group (PC), and 100 and 200 mg kg-1 GOS-administered groups (GL and GH, respectively). GOS administration significantly improved epidermal thickness and decreased CD4+ cell numbers. The concentrations of IgE, OVA-specific IgE, and inflammatory cytokines (IL-4, IL-5, and INF-γ) in GH group mice were significantly lower than those in OVA-C group mice. Firmicutes and Bacteroidetes were identified as the major phyla in the intestinal microbiota in mice, and the relative abundance of Deferribacteres was significantly lower in the GH group than in the OVA-C group. Deferribacteraceae and Mucispirillum species were significantly lower in the GH group than in the OVA-C group. The relative abundance of Muribaculum species was significantly lower, but those of Lachnospira and Lactococcus species were significantly higher in the GH group than in the OVA-C group. Our results suggest that the alleviation effect of GOS on allergic dermatitis induced by OVA sensitization was achieved by regulating hypersensitive immune responses by improving the intestinal microbial ecosystem.