Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. Multiepitope T-cell vaccines are more likely to generate a broad long-lasting immune response than those composed of single epitopes. We recently reported a novel multivalent cytotoxic T-lymphocyte peptide construct derived from the Tax protein of HTLV-1 separated by arginine spacers that elicited high cellular responses against individual epitopes simultaneously in human leukocyte antigen (HLA)-A*0201 transgenic mice. We now report the effect of epitope orientation on the processing of the multiepitope construct by 20s proteasomes and the effect of the processing rates on the immunogenicity of the intended epitopes. A positive correlation was found between processing rates and the immunogenicity of the intended epitopes. The construct with the highest immunogenicity for each epitope was tested for protective efficacy in a preclinical model of infection using HTLV-1 Tax recombinant vaccinia virus and HLA-A*0201 transgenic mice. Mice vaccinated with the multiepitope construct displayed a statistically significant reduction in viral replication that was dependent on CD8 T cells. Reduction in viral replication was also confirmed to be specific to Tax-vaccinia virus. These results demonstrate the activation of Tax-specific CD8+ T cells by vaccination and are supportive of a multivalent peptide vaccine approach against HTLV-1 infections.