Research to enhance the efficiency of vaccines focuses mainly on improving either the adjuvant or the type and form of the antigen. This study evaluates the influence of the administration route on the efficiency of a peptide‐based vaccine. Peptide vaccines are generally administered subcutaneously or intradermally, from where they must reach secondary lymphatic organs to induce an immune response. We analyzed the efficacy of peptide vaccines administered directly into a lymph node. Using a MHC class I‐binding peptide from lymphocytic choriomeningitis virus, we found that intralymphatic injection enhanced immunogenicity by as much as 10⁶ times when compared to subcutaneous and intradermal vaccination. Intralymphatic administration induced CD8 T cell responses with strong cytotoxic activity and IFN‐γ production that conferred long‐term protection against viral infections and tumors. These results should have immediate implications for clinical immunotherapy of infectious disease and cancer.