Strategies to manipulate the intestinal microbiota have been considered to promote immune health. The aim of the present study was to examine whether fructo-oligosaccharide, a typical prebiotic, could suppress antigen-specific skin inflammation by favourably changing the population of intestinal microbiota. Female BALB/c mice were fed a synthetic diet with or without fructo-oligosaccharide supplementation for 3 weeks and were then epicutaneously immunised with 2,4-dinitrofluorobenzene. Afterwards, mice continued to receive their respective diets. At 5 d after immunisation, the mice were ear challenged with the hapten. Ear swelling after the challenge was significantly reduced in the mice fed the diet supplemented with fructo-oligosaccharide than in mice fed the control diet. To characterise the change in the intestinal microbiota, DNA samples isolated from fresh faeces were subjected to PCR-denaturing gradient gel electrophoresis and real-time PCR based on 16S rDNA gene sequences. Dietary fructo-oligosaccharide altered the composition of intestinal microbiota. The numbers of bifidobacteria, but not lactobacilli, were significantly higher in mice fed the fructo-oligosaccharide-supplemented diet than in mice fed the control diet. Ear swelling was negatively correlated with the numbers of bifidobacteria in the faeces. Sequence analysis revealed that Bifidobacterium pseudolongum was the most predominant bifidobacteria in the intestine of mice fed the fructo-oligosaccharide-supplemented diet. These results suggest that consumption of fructo-oligosaccharide reduces contact hypersensitivity, which is associated with proliferation of B. pseudolongum in the intestinal tract of mice.