Allergic asthma is an increasing burden in industrialized countries, where treatments are limited to treating symptoms rather than root causes. In recent years, the use of inhaled probiotic cocktails has shown promise in preventing allergic asthma. However, the mechanisms by which such probiotics prevent sensitization remain to be elucidated. In this study, we focused on exopolysaccharide (EPS) from B. subtilis, a ubiquitous soil bacterium. We found that intratracheal EPS treatment prior to sensitization with house dust mite (HDM) limited lung mucus hypersecretion and eosinophilia. Furthermore, EPS treatment inhibited accumulation of TH2, but not TH1 or TH17 subsets. To determine whether EPS was affecting TH2 cell priming by dendritic cells (DCs), we compared the composition and phenotype of DCs in the lung and lung-draining lymph node (LDLN) after EPS and HDM treatment. Surprisingly, pre-treatment with EPS induced DC activation, as measured by an increase in CD86, and a decrease in the ability to uptake HDM and migrate to the LDLN. Additionally, EPS-pretreated BMDCs were unable to induce antigen-specific T cell proliferation in vitro. Transcriptionally, EPS downregulated proteins associated with DC induction of T cell responses such as innate cytokines, costimulatory molecules, and chemokines. Together, our data suggest that EPS inhibits allergic sensitization by dysregulating DCs prior to HDM exposure, thereby leading to decreased TH2 priming and allergic inflammation. Thus, we provide a potential mechanism by which inhalation of a single prebiotic molecule can prevent allergic sensitization.