Background: TAS0313 is a cancer vaccine cocktail comprising three long peptides with a total of 12 cytotoxic T lymphocyte epitope peptides. We performed a multicenter phase I/II study including patient (pts) with urothelial carcinoma (UC) treated using TAS0313 combined with pembrolizumab. Methods: The enrolled pts with a histologically or cytologically confirmed diagnosis of urothelial carcinoma 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. For cohort C1, eligible pts were those who received platinum-based chemotherapy and were naïve to immune checkpoint inhibitors (ICI). For cohort C2, eligible pts were those who progressed onto treatment with pembrolizumab. TAS0313 (9 mg) 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, while pembrolizumab (200 mg) was intravenously administered on day 1 of cycle 1 or later in 21-day cycles until disease progression or unacceptable toxicity occurred. Tumor response was evaluated using the RECIST v1.1 criteria. The TAS0313 target antigen-specific immunoglobulin G (IgG) was analyzed before and after treatment. The primary objective was to evaluate efficacy, while the secondary objective was to evaluate the safety and tolerability of the combination therapy. Results: As of 10th September 2020, 46 pts with a median age of 71.0 and 65.5 years, in cohort C1 (n = 36) and cohort C2 (n = 10), respectively, have been treated with the combination therapy. For cohorts C1 and C2, the median follow-up duration was 6.47 and 6.95 months, while the median treatment duration was 4.86 and 2.56 months, respectively. In cohort C1, the overall response rate and disease control rate (DCR) were 33.3% (16.7% complete response, 16.7% partial response) and 66.7% (33.3% stable disease), respectively. The median progression-free survival was 5.0 months, median overall survival (OS) was not yet reached, and 1-year OS rate was 74.3%. The best overall response in cohort C2 was stable disease in 5/10 pts, resulting in a DCR of 50.0%. Increase in IgG level was detected after treatment in both the cohorts. The most common adverse drug reactions (ADRs) of TAS0313 and/or pembrolizumab were grade 1–2 injection site reactions and pyrexia. There were no grade 3–5 ADRs with an incidence of ≥10%. Conclusions: This study confirmed the tolerability, safety, and immune response of TAS0313 combined with pembrolizumab in cohorts C1 and C2. We observed promising efficacy in pts with ICI-naïve UC in cohort C1; however, in pts with pembrolizumab-refractory UC in cohort C2, limited efficacy was seen. Therefore, a large-scale randomized study is needed to clarify the benefits of TAS0313 combined with ICI in ICI-naïve pts. Clinical trial information: 183824.