Background: Food allergies (FAs) are associated with alterations in the gut microbiota, epithelial barrier and immune tolerance. These dysfunctions are observed in the first month of life, revealing that early intervention is crucial for disease prevention. Nutritional strategies such as prebiotics may reduce FAs in children. Indeed some prebiotics such as galacto-oligosaccharides (GOS) and inulin are able to induce tolerance, epithelial barrier reinforcementand gut microbiota modulation, but the ideal period for intervention is unknown. Herein, we investigated whether GOS/inulin supplementation during gestation could protect progeny against FAs in mice. Methods: The mothers received a control diet or an enriched diet with GOS/inulin exclusively during the pregnancy. At the weaning, pups were intraperitoneally sensitized and orally challenged with a wheat allergen. After the challenge pups symptoms were evaluated and we analyzed allergic and tolerogenic parameters. Moreover, mothers and pups fecal microbiota and short chain fatty acids (SCFAs) were analyzed throughout the protocol. Results: We demonstrated that prebiotics supplementation induced a strong restructuration of the fecal microbiota of mice toward beneficial strains during gestation and partially during mid-lactation. This specific microbiota was transferred to pups and maintained to adulthood. Moreover, B and T regulator subsets were increased in pups born from supplemented mothers, inducing a tolerogenic environment and protecting them against FAs. Conclusions: Our study demonstrates that prebiotics supplementation during pregnancy induces on the offspring a tolerogenic environment and a microbial imprint, leading to a reduction of FA development.