Intramuscular immunization of mice with DNA cocktail vaccines, comprising potential protective antigens P36, P46, NrdF, and P97or P97R1 of Mycoplasma hyopneumoniae, induced strong Th1-polarized immune responses against each antigen, with only P46 eliciting a serum IgG response. Subcutaneous immunization with protein cocktail vaccines, surprisingly, induced both Th1-polarized immune response as well as antibody response whereas mice immunized with DNA cocktail vaccines followed by boosting with protein cocktail vaccines generated strong Th1-polarized and humoral immune responses. P97 was not recognized by serum antibodies from commercial bacterin-immunized mice indicating potential lack of expression of this important antigen in inactivated whole-cell vaccines.