Background: Potent helper action is necessary for peptide-based vaccines to efficiently induce antitumor immune responses against advanced cancer. A clinical trial for advanced esophageal squamous cell carcinoma was carried out for patients with HLA-A*2402 using epitope peptides derived from novel cancer-testis antigens, LY6K and TTK, in combination with CpG-B. Methods: This study investigated the feasibility and the toxicity as well as induction of tumor antigen-specific immune responses. In phase I trial, nine patients were vaccinated on days 1, 8, 15, and 22 of each 28-day treatment cycle with peptide LY6K-177, peptide TTK-567, and CpG-B (level-1; 0, level-2; 0.02, level-3; 0.1 mg/kg) . In phase II, eight patiens were vaccinaed the same pepides on the same schedule wih CpG-B (0.1ng/kg). Results: All were tolerated by this treatment. In phase I, LY6K-specific T cell responses in PBMCs were detected in two of the three patients in each level. In particular, two patients in level-2/3 showed potent LY6K-specific T cell responses. In contrast, only two patients in level-2/3 showed TTK-567-specific T cell responses. The frequency of LY6K-177 or TTK-567-specific CD8+ T cells increased in patients in level-2/3 (with CpG). The vaccination with peptides and CpG-B increased and activated both plasmacytoid dentritic cells and natural killer cells, and increased the serum level of a-interferon. There were no complete response (CR) and partial response (PR), however, one of three patients in level-1, and four of six patients in level-2/3 showed stable disease (SD). This vaccine protocol is therefore recommended to undergo further phase II trials. In phase II, there were no CR and PR. LY6K and TTK-specific T cell responses were observed wihin 8 weeks and the frequency of the antigens specific CTL was increased after continuous vaccination. The medican survival time (MST) of all vaccination patients was 194 days. Conclusions: Vaccination with LY6K-177 and TTK-567 in combination with CpG-B successfully elicited antigen-specific CD8+ T cell responses and enhanced the innate imunity of patients with advanced esophageal squamous cell carcinoma. Clinical trial information: NCT00669292