Dendritic cells (DCs) are highly efficient antigen-presenting cells capable of priming both cytotoxic and helper T cells in vivo. Recent studies have demonstrated the potential use of DCs that are modified to carry tumor-specific antigens in cancer vaccines. However, the optimal administration route of DC-based vaccines to generate the greatest anti-tumor effect remains to be determined. This study is aimed at comparing the levels of immune responses and anti-tumor effect generated through different administration routes of DC-based vaccination. We chose the E7 gene product of human papillomavirus (HPV) as the model antigen and generated a stable DC line (designated as DC-E7) that constitutively expresses the E7 gene. Among the three different routes of DC-E7 vaccine administration in a murine model, we found that intramuscular administration generated the greatest anti-tumor immunity compared with subcutaneous and intravenous routes of administration. Furthermore, intramuscular administration of DC-E7 elicited the highest levels of E7-specific antibody and greatest numbers of E7-specific CD4+ T helper and CD8+ T cell precursors. Our results indicate that the potency of DC-based vaccines depends on the specific route of administration and that intramuscular administration of E7-transfected DCs generates the most potent E7-specific anti-tumor immunity.