Intramuscular administration of DNA vaccines can lead to the generation of antigen-specific immune responses through cross-priming mechanisms. We propose a strategy that is capable of leading to local inflammation and enhancing cross-priming, thus resulting in improved antigen-specific immune responses. Therefore, in this study, we evaluated the immunological responses elicited through electroporation-mediated intramuscular administration of a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 E7 (CRT-E7) in combination with DNA expressing HLA-A2 as compared with CRT-E7 DNA vaccination alone. We found that the co-administration of a DNA vaccine in conjunction with a DNA encoding a xenogenic major histocompatibility complex (MHC) molecule could significantly enhance the E7-specific CD8+ T-cell immune responses and antitumor effects against an E7-expressing tumor, TC-1, in C57BL/6 tumor-bearing mice. Furthermore, a similar enhancement in E7-specific immune responses was observed by the co-administration of CRT-E7 DNA with DNA encoding other types of xenogenic MHC class-I molecules. This strategy was also applicable to another antigenic system, ovalbumin. Further characterization of the injection site revealed that the co-administration of HLA-A2 DNA led to a significant increase in the number of infiltrating CD8+ T lymphocytes and CD11b/c+ antigen-presenting cells. Furthermore, the E7-specific immune responses generated by intramuscular co-administration of CRT-E7 with HLA-A2 DNA were reduced in HLA-A2 transgenic mice. Thus, our data suggest that intramuscular co-administration of DNA encoding xenogenic MHC class-I can further improve the antigen-specific immune responses, as well as antitumor effects generated by DNA vaccines through enhancement of cross-priming mechanisms.