Milk glycans play key roles in shaping and maintaining a healthy infant gut microbiota. Core fucosylation catalyzed by fucosyltransferase (Fut8) is the major glycosylation pattern on human milk N-glycan, which was crucial for promoting the colonization and dominant growth of Bifidobacterium and Lactobacillus spp. in neonates. However, the influence of core-fucose in breast milk on the establishment of early-life immune tolerance remains poorly characterized. In this study, we found that the deficiency of core-fucose in the milk of maternal mice caused by Fut8 gene heterozygosity (Fut8+/-) resulted in poor immune tolerance towards the ovalbumin (OVA) challenge, accompanied by a reduced proportion of intestinal RORγt+ Treg cells and the abundance of Lactobacillus spp., especially L. reuteri and L. johnsonii, in their breast-fed neonates. The administration of the L. reuteri and L. johnsonii mixture to neonatal mice compromised the OVA-induced allergy and up-regulated the intestinal RORγt+ Treg cell proportions. However, Lactobacillus mixture supplementation did not alleviate allergic responses in RORγt+ Treg cell-deficient mice caused by Rorc gene heterozygosity (Rorc+/-) post OVA challenge, indicating that the intervention effects depend on the RORγt+ Treg cells. Interestingly, instead of L. reuteri and L. johnsonii, we found that the relative abundance of another Lactobacillus spp., L. murinus, in the gut of the offspring mice was significantly promoted by intervention, which showed enhancing effects on the proliferation of splenic and intestinal RORγt+ Treg cells in in vitro studies. The above results indicate that core fucosylation of breast milk N-glycans is beneficial for the establishment of RORγt+ Treg cell mediated early-life immune tolerance through the manipulation of symbiotic bacteria in mice.