Background: TAS0313 is a cancer vaccine cocktail comprising three long peptides with a total of 12 cytotoxic T lymphocyte (CTL) epitope peptides. These peptides were derived from eight cancer-associated antigens (EGFR, KUA, LCK, MRP3, PTHRP, SART2, SART3, and WHSC2) that are overexpressed in various cancer types including glioblastoma (GBM). We performed a single-arm, multicenter efficacy confirmatory cohort study as part of the TAS0313 Phase I/II study in patients with recurrent GBM. Methods: The enrolled patients with histologically or cytologically confirmed grade IV GBM (including gliosarcoma, giant cell glioblastoma, and epithelioid glioblastoma) had at least one of the following HLA types: HLA-A*02:01, -A*02:06, -A*02:07, -A*11:01, -A*24:02, -A*31:01, or -A*33:03. Eligible patients were those who received standard radiotherapy and temozolomide, had a confirmed first or second recurrence, or progression with measurable disease outcomes with good performance status (Karnofsky Performance Status score > 70). TAS0313 (27mg) was subcutaneously administered on days 1, 8, and 15 of cycles 1 and 2 and day 1 of cycle 3 or later in 21-day cycles until disease progression or unacceptable toxicity occurred. Tumor response was evaluated using The Response Assessment in Neuro-Oncology criteria. The antigen-specific CTL and immunoglobulin G (IgG) were analyzed by ELISPOT assay and Luminex assay, respectively, before and after treatment. The primary objective was to evaluate the efficacy of TAS0313. Secondary and exploratory methods were used to evaluate the safety and immune response of TAS0313. Results: As of 10th September 2020, 10 patients have been treated with TAS0313 (eight with GBM and two with gliosarcoma). The median age of participants was 56.5 [range, 33–69 years], and methylation status of the MGMT promoter was methylated in four patients, unmethylated in four, and unknown in two. The best overall response was partial response 1/10 (10.0%) and stable disease 4/10 (40.0%). One case showed 69.1% tumor shrinkage. The treatment of two patients was ongoing for over 7 months. The 6-month progression-free survival (PFS) rate was 25.0%, and the median PFS was 2.3 months. The most common adverse drug reactions (ADRs) were grade 1–2 injection site reactions and pyrexia. There were no grade 4 or 5 ADRs. In some patients, TAS0313 treatment was confirmed to increase CTL and IgG levels compared with pre-treatment samples. Conclusions: TAS0313 showed promising efficacy with expected immune responses and favorable safety and tolerability in patients with recurrent GBM. Further investigation of TAS0313 is warranted to validate our findings. Clinical trial information: JapicCTI-183824.