In the early stage of this study, three strains of Lactobacillus with anti-soybean allergy potential were screened: Lactobacillus acidophilus CICC 6081, Lactobacillus delbrueckii subsp. Bulgaricus CICC 6103 and Lactobacillus plantarum subsp. Plantarum CICC 20988. The aim of this study was to analyze the desensitization effect of three strains of Lactobacillus administered by gavage to soybean-allergic mice through the differentiation of immune cells in intestinal lymph nodes and the changes to gut microbiota. The results showed that the three strains of Lactobacillus could stimulate the proliferation of dendritic cells (DCs) and regulate the balance of Th1/Th2 differentiation in the MLNs and PPs of soybean-allergic mice. Furthermore, the Th17/Tregs cell-differentiation ratio in the MLNs of the Lactobacillus-treated mice was significantly lower than that of the allergic mice (p < 0.05). Compared to the control group, the Shannon, Sobs and Ace indexes of intestinal microbiota in the allergic mice were significantly increased (p < 0.05), and the proportion of Clostridiales was significantly higher (p < 0.05), which was reversed by Lactobacillus gavage. In conclusion, the three strains of Lactobacillus can inhibit the intestinal mucosal immune response and regulate gut microbiota balance in soybean-allergic mice.