: Background: Prebiotic galacto‐ and fructo‐oligosaccharides (scGOS/lcFOS) resembling non‐digestible oligosaccharides in human milk reduce the development of atopic disorders. However, the underlying mechanisms are still unclear. Galectins are soluble‐type lectins recognizing β‐galactoside containing glycans. Galectin‐9 has been shown to regulate mast cell degranulation and T‐cell differentiation. In this study, the involvement of galectin‐9 as a mechanism by which scGOS/lcFOS in combination with Bifidobacterium breve M‐16V protects against acute allergic symptoms was investigated. Methods: Mice were sensitized orally to whey, while being fed with a diet containing scGOS/lcFOS and Bifidobacterium breve M‐16V (GF/Bb) or a control diet. Galectin‐9 expression was determined by immunohistochemistry in the intestine and measured in the serum by ELISA. T‐cell differentiation was investigated in the mesenteric lymph nodes (MLN) as well as in galectin‐9‐exposed peripheral blood mononuclear cells (PBMC) cultures. Sera of the mice were evaluated for the capacity to suppress mast cell degranulation using a RBL‐2H3 degranulation assay. In addition, in a double‐blind, placebo‐controlled multicenter trial, galectin‐9 levels were measured in the sera of 90 infants with atopic dermatitis who received hydrolyzed formulae with or without GF/Bb. Results: Galectin‐9 expression by intestinal epithelial cells and serum galectin‐9 levels were increased in mice and humans following dietary intervention with GF/Bb and correlated with reduced acute allergic skin reaction and mast cell degranulation. In addition, GF/Bb enhanced Th1‐ and Treg‐cell differentiation in MLN and in PBMC cultures exposed to galectin‐9. Conclusions: Dietary supplementation with GF/Bb enhances serum galectin‐9 levels, which associates with the prevention of allergic symptoms.