Serine protease inhibitor 6 (SPI-6), also called Serpinb9, inhibits granzyme B and thus may provide a method for delaying apoptotic cell death in dendritic cells. We have previously enhanced DNA vaccine potency by targeting antigen to MHC antigen presentation pathways, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, domain II of Pseudomonas aeruginosa exotoxin A, or the sorting signal of the lysosome-associated membrane protein type 1. In this study, we explored intradermal coadministration of DNA encoding SPI-6 with DNA constructs encoding human papillomavirus type 16 E7 linked to these intracellular targeting molecules for its ability to generate E7-specific CD8+ T-cell immune responses and E7-specific antitumor effects. This combination of strategies resulted in significantly increased E7-specific CD8+ T-cell and CD4+ Th1-cell responses, enhanced tumor treatment ability, and stronger tumor protection when compared with vaccination without SPI-6. Among these targeting strategies tested, mice vaccinated with Sig/E7/lysosome-associated membrane protein type 1 mixed with SPI-6 showed the greatest fold increase in E7-specific CD8+ T cells ( approximately 5-fold). Vaccination with a nonfunctional mutant of SPI-6 did not result in immune enhancement, indicating that enhancement was dependent on the antiapoptotic function of SPI-6. Our results suggest that DNA vaccines combining strategies that enhance MHC class I and II antigen processing with SPI-6 have potential clinical implications for control of viral infection and neoplasia.