We report the long-term outcome on phase I study of combined WT1 peptide vaccination/temozolomide against newly diagnosed glioblastomas. Immunological biomarker and response were investigated in trials targeting newly diagnosed and recurrent cases. The survival analysis was performed on 7 patients in the phase I study. Peripheral blood mononuclear cells were obtained from 7 patients as well as 30 patients who had WT1 vaccination alone against the recurrent glioblastomas. FACS analyses were conduced on total of 37 samples; WT1-specific T cells were indentified using tetramer assay and sub-classified with CCR7 and CD45RA expression. The results were compared between responder and non-responder group. Median PFS of the 7 patients were 43.5 months and 5 patients showed OS longer than 3 years. In responder group, the frequencies of CD4 + central memory (p < 0.01), CD8 + effector memory (EM) (p < 0.01) and WT1-specific EM (p < 0.05) were higher statistically significantly before the vaccination, and those of CD4 + effector (E) (p < 0.01), CD8 + E (p < 0.05) and WT1-specific E (p < 0.01) were lower. The frequencies of WT1-specific T cell (p < 0.001) and WT1-specific EM (p < 0.001) significantly increased after vaccination. Biomarkers were identified, as well as possible immunological response that might present a proof of concept.