[Objective] Aim of this study was to investigate the effect of whole peptidoglycan (WPG) from Lactobacillus acidophilus on regulating Th1/Th2 and Treg/Th17 balance in sensitized splenocytes. [Methods] BALB/c mice were intraperitoneally injected with β-Lactoglobulin (β-Lg), the sensitized splenocytes were stimulated in vitro with different doses of WPG. Supernatants were collected and measured total IgE and β-Lg-specific IgE, Th1/Th2 and Treg/Th17 related cytokines (IFN-γ, IL-4, TGF-β, IL-17) production by ELISA assay; the percentage of CD3+, CD4+, CD8+ subgroups in spleen cells were measured by flow cytometry, and the levels of T-bet, GATA-3, Foxp3 and RORγt mRNA were measured by RT-PCR. [Results] Compared with the allergy group, treatment with medium and high dose of WPG significantly decreased IL-17, RORγt mRNA response. In addition, the numbers of CD3+ and CD4+ cells and the ratio of the secretion of total IgE and β-Lg-specific IgE, Th2-type (IL-4, GATA-3 mRNA) and Th17-type (CD4+/CD8+), Th1-type (IFN-γ, T-bet mRNA) and Treg-type (TGF-β, Foxp3 mRNA) response were increased in the splenocytes stimulated with WPG in a dose manner. [Conclusion] Treatment with WPG from Lactobacillus acidophilus could regulate the imbalance of Th1/Th2 and Treg/Th17 in allergic spleen cells.