Millions of women are currently infected with high-risk human papillomavirus (HPV), which is considered to be a major risk factor for cervical cancer. Thus, it is urgent to develop therapeutic vaccines to eliminate the established infections or HPV-related diseases. In the present study, using the mycobacterium tuberculosis heat shock protein 70 (MtHSP70) gene linked to the modified HPV 16 E7 (mE7) gene, we generated two potential therapeutic HPV DNA vaccines, mE7/MtHSP70 and SigmE7/MtHSP70, the latter was linked to the signal peptide gene sequence of human CD33 at the upstream of the fusion gene. We found that vaccination with the mE7/MtHSP70 DNA vaccine induced a stronger E7-specific CD8+ T cell response and resulted in a more significant therapeutic effect against E7-expressing tumor cells in mice. Our results demonstrated that HSP70 can play a more important role in mE7 and MtHSP70 fusion DNA vaccine without the help of a signal peptide. This may facilitate the use of HSP70 and serve as a significant reference for future study.