Asthma is a long-lasting lung disease that causes breathing problems. 50 to 80 % of asthma cases may be caused by dust mites and their droppings. This severe reaction is initiated by the allergen-specific T cell responsiveness. Effective antigen-specific treatments or preventions are still lacking. Using a murine asthma model induced by dust mite extract, we first report that co-immunization of DNA and protein vaccines encoding the dust mite specific antigen (Der p1) can prevent allergic responses, including the antigen specific IgE level and infiltration of lymphocytes and mast cells to the site of the allergen challenge. The prevention of Der p1induced asthma is directly related to the induction of a population of Der p1 specific iTreg cells exhibiting a CD4+/CD25-/FoxP3+ phenotype. This subtype of iTreg cells express IL-10 and TGF-beta, but not CTLA-4 and CCR7 which are well-known markers of natural nTreg cells. Adoptive transfer of the iTreg cells is also able to suppress the Der p1-induced asthma, suggesting the inhibition potency of the iTreg cells directly on the asthma causing T cells. The study demonstrates that utility of the co-immunization strategy may present a prophylactive approach clinically and offer us a novel strategy to prevent other allergic and asthma diseases in human and animals.