Background: In a murine model of allergic inflammation, Bifidobacterium breve M-16V has been shown to reduce IL-4 and IgE by inducing IL-10 and IFN-γ. However, it remains unknown whether this strain has the same effect in humans with allergic disease. Objective: To determine the effects of Bifidobacterium breve M-16V combined with a prebiotic oligosaccharide mixture (synbiotic) on atopic markers, ex vivo cytokine production by peripheral blood mononuclear cells (PBMCs) and circulating regulatory T cell percentage in infants with atopic dermatitis. Methods: In a double-blind, placebo-controlled multi-centre trial, 90 infants with atopic dermatitis, age <7 months, were randomized to receive an infant formula with Bifidobacterium breve M-16V and a mixture of short chain galactooligosaccharides and long chain fructooligosaccharides (Immunofortis ®), or the same formula without synbiotics during 12 weeks. At week 0 and 12, plasma levels of IL-5, IgG1, IgG4, CTACK and TARC, ex vivo cytokine responses by PBMCs and percentage of regulatory T cells, were determined. Results: There were no significant differences between the synbiotic and the placebo group in IL-5, IgG1, IgG4, CTACK and TARC levels and ex vivo cytokine production by anti-CD3/anti-CD28-stimulated PBMCs. With allergen-specific stimuli, we found a decreased IL-12p40/70 and IL-12p70 production in response to egg allergen (P = 0.04 and P = 0.01, respectively) and decreased IL-12p70 production in response to peanut allergen (P = 0.003) in the synbiotic compared with the placebo group. Circulating regulatory T cell percentage did not significantly differ between the groups. Conclusions and Clinical Relevance: This synbiotic mixture has no detectable effect on plasma levels of the analysed atopic disease markers, ex vivo cytokine production and circulating regulatory T cell percentage in infants with atopic dermatitis, besides down-regulation of IL-12 production in egg- and peanut-stimulated PBMCs. These results do not support the use of this synbiotic in clinical practice.