Although antigen-specific immune responses including cytotoxic T cells (CTLs) against antigen peptide could be enhanced after tumor antigen peptide vaccinations, the immune responses do not necessarily result in a decrease or eradication of tumor cells in the vaccination trials. We focused on whether antigen-specific CTLs could be damaged by the repeated stimulation of antigenic peptide and whether regulatory T (Treg) cells would be increased by the administration of WT1 peptide. We administered WT1 peptide 22 times over 18 months in a CML patient who was being treated with imatinib. Although WT1 peptide administration every 2 weeks did not show any beneficial effects on the minimal residual disease (copies of bcr-abl transcripts), the transcripts remarkably decreased to the level of major molecular response after changing the administration interval of WT1 peptide from 2 to 4 weeks. An ex vivo study demonstrated that re-stimulation with WT1 peptide made WT1-specific T cells less reactive to WT1 tetramers and the impaired reactivity of CTLs lasted at least for 1 week. In addition, the cytotoxicity of the T cells was hampered by re-stimulation. Treg cells increased up to more than fivefold at the end of the WT1 administration period. The present findings suggested that the administration of the peptide every 4 weeks is superior to every 2 weeks. In addition, the findings that Treg cells increased gradually in accordance with the duration of WT1 peptide administration revealed the significance of manipulating Treg cells for establishing an efficient tumor antigen peptide vaccination.