Infection with human papillomavirus, particularly type 16 (HPV16), is highly associated with the development of cervical intraepithelial neoplasia and cervical cancer. The two early viral oncogenes, E6 and E7, are selectively retained and constitutively expressed in tumor cells and are therefore attractive immunotherapeutic targets. Thus a vaccine strategy based on recombinant HPV16 E6/E7 fusion protein represents an efficient approach against HPV16-associated tumors. Although the expression level of HPV16 E6/E7 fusion protein was presumed to be low, direct experimental proof in vivo was lacking. To enhance the expression level and investigate its antitumor efficacy in vivo, we constructed a modified HPV16 E6/E7 fusion gene with three point-mutations and expressed it in Escherichia coli. The encoded protein, denoted mE6(1-120)/mE7(1-60), comprises 120 N-terminus amino acids of E6 and 60 N-terminus amino acids of E7 plus a histine tag, was purified on an affinity column, and subsequently characterized by Western blotting. Immunization of mice with mE6(1-120)/mE7(1-60) completely protected them against subsequent challenge and rechallenge with TC-1 tumor cells expressing HPV16 E6 and E7 proteins. In the therapeutic experiments, most mice eliminated the preexisting tumors and had a long-term protection. Consistent with the results of in vivo experiments, the splenocytes from immunized mice elicited cytotoxic T lymphocytes and specifically lysed TC-1 cells in vitro. More importantly, the expression level of mE6(1-120)/mE7(1-60) was significantly improved, meeting the necessary quantity required for a vaccine clinical trial. In conclusion, these data provide a scientific basis for the use of modified mE6(1-120)/mE7(1-60) in future human trials.