Dietary intervention with a unique prebiotic nondigestible carbohydrate mixture has been shown to reduce the development of allergic disease in infants at risk. In this study, the involvement of CD25(+) regulatory T-cells (Treg) in the carbohydrate-induced effects was investigated in mice orally sensitized with whey using adoptive transfer experiments. Donor mice were sensitized with whey and fed a diet containing short-chain galacto-, long-chain fructo- and acidic-oligosaccharides, or a control diet starting 2 wk before sensitization. The acute allergic skin reaction upon intradermal whey challenge was determined and whey-specific Ig were measured. Splenocytes of the donor mice were transferred to naïve recipient mice after partial ex vivo depletion of CD25(+) Treg. The prebiotic diet clearly diminished the acute allergic skin reaction (P < 0.001). Whey-sensitized recipient mice transferred with splenocytes from whey-sensitized, prebiotic-fed donor mice displayed almost complete prevention of the acute allergic skin reaction compared with mice receiving cells from sham-sensitized, prebiotic-fed donor mice (P < 0.001). Partial depletion of CD25(+) T-cells inhibited these effects (P < 0.001), although IgE sensitization was not prevented. This study indicates the involvement of whey-specific CD25(+) Treg in the suppression of the allergic effector response induced by dietary intervention with prebiotics.