Human papillomavirus, particularly type 16 (HPV16) is present in more than 99% of cervical cancers. E7 is the major oncogenic protein produced in cervical cancer-associated HPV16. An efficient vaccine against viral infection requires induction of strong humoral and cellular responses against viral proteins. Heat shock proteins (HSPs) like Gp96 have been described as potent tumor vaccines in animal models and are currently studied in human clinical trials. In this study, we investigated the utility of HPV16 E7 along with Gp96 as an adjuvant in C57BL/6 mice model. We compared the level of humoral and cellular immune responses by E7+Gp96 co-injection as DNA/DNA and prime-boost (DNA/protein) immunization strategies. In prime-boost immunization strategies, we first immunized C57BL/6 mice with the complete open-reading frame of E7 and Gp96 (pcDNA-E7 and pcDNA-Gp96) and then boosted with rE7, rNT-gp96 (N-terminal extension of Gp96) and rCT-gp96 (C-terminal extension of Gp96) mixed with Montanide 720 in different formulations. The humoral immune responses against rE7 and the different truncated forms of rGp96 suggested a mixed Th1/Th2 response with high intensity toward Th2. Assessment of lymphoproliferative and cytokine responses against rE7 and the different fragments of Gp96, showed that DNA vaccination including E7 and Gp96 induced Th1 response. We concluded that co-delivery of naked DNA E7+Gp96 plasmid was immunologically more effective than E7 alone. Our study demonstrated that co-delivery of E7+Gp96 as DNA/DNA and E7+CT-gp96 as DNA/protein could be an effective approach to induce E7-specific immune responses as a potential vaccine candidate for cervical cancer.