Background: Transcutaneous immunization (TCI) approaches utilize skin associated lymphatic tissues to elicit specific immune responses. In this context, the imidazoquinoline derivative imiquimod formulated in Aldara applied onto intact skin together with a cytotoxic T lymphocyte (CTL) epitope induces potent CTL responses. However, the feasibility and efficacy of the commercial imiquimod formulation Aldara is limited by its physicochemical properties as well as its immunogenicity. Methodology/Principal Findings: To overcome these obstacles, we developed an imiquimod-containing emulsion gel (IMI- Gel) and characterized it in comparison to Aldara for rheological properties and in vitro mouse skin permeation in a Franz diffusion cell system. Imiquimod was readily released from Aldara, while IMI-Gel showed markedly decreased drug release. Nevertheless, comparing vaccination potency of Aldara or IMI-Gel-based TCI in C57BL/6 mice against the model cytotoxic T- lymphocyte epitope SIINFEKL, we found that IMI-Gel was equally effective in terms of the frequency of peptide-specific T- cells and in vivo cytolytic activity. Importantly, transcutaneous delivery of IMI-Gel for vaccination was clearly superior to the subcutaneous or oral route of administration. Finally, IMI-Gel based TCI was at least equally effective compared to Aldara- based TCI in rejection of established SIINFEKL-expressing E.G7 tumors in a therapeutic setup indicated by enhanced tumor rejection and survival. Conclusion/Significance: In summary, we developed a novel imiquimod formulation with feasible pharmaceutical properties and immunological efficacy that fosters the rational design of a next generation transcutaneous vaccination platform suitable for the treatment of cancer or persistent virus infections.