Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia, a chronic nonfatal disease affecting pigs of all ages. The goal of this study was to design DNA vaccines by constructing plasmid pcDNA3/P42, carrying the heat shock protein gene P42 of M. hyopneumoniae, and to evaluate the immune responses elicited in BALB/c mice. The expression of P42 was first examined in transfected NIH 3T3 cells by reverse transcription-PCR to ensure that the construct was functional. The humoral and cell-mediated immune responses induced by the plasmid were further evaluated in BALB/c mice through intramuscular injection. Both immunoglobulin G1 (IgG1) and IgG2a levels were 64 times those of the control groups during the first 8 weeks. The levels of interleukin-2 (IL-2), IL-4, and gamma interferon mRNAs in the immunized animals were elevated, and the proliferation of spleen cells was also enhanced in the immunized animals. The results indicate that pcDNA3/P42 DNA immunization induces both Th1 and Th2 immune responses. In addition, antiserum from the immunized animals was found to inhibit the growth of M. hyopneumoniae. The present study reveals that DNA vaccination could be a new strategy against infection by M. hyopneumoniae and may have potential for developing vaccines for other infectious diseases as well.