Background: Since a phase I cancer vaccination trial using three novel HLA-A24-binding peptides derived from Oncoantigen, TTK protein kinase (TTK), lymphocyte antigen 6 complex locus K (LY6K), and IGF-II mRNA binding protein 3 (IMP-3), had revealed safety and immunogenicity in advanced esophageal squamous cell carcinoma (ESCC), we further performed a phase II trial to evaluate the survival benefit of the cancer vaccination. Methods: Sixty advanced ESCC patients who failed to standard therapy were enrolled to evaluate OS, PFS, immunological response employing ELISpot and pentamer assay, and adverse effect. Each of the three peptides (1 mg each) was subcutaneously administered with 1 ml of IFA weekly for five weeks. All enrolled patients had received the vaccination without knowing HLA-A status double-blindly, and the HLA genotype were key-opened at analysis point and then, the endpoints were evaluated between HLA-A*2402 positive (24(+), n=35) and HLA-A*2402 negative (24(-), n=25) group. Results: The treatment was well-tolerated without any treatment-associated adverse events of grade 3 or 4. The patient backgrounds did not differ between the 24(+) and 24(-) group. Although the difference in OS after the vaccination between the 24(+) and 24(-) group did not reach a significant level (4.6 vs. 2.6 month, respectively, p=0.136), the PFS in the 24(+) group was significantly better than that in the 24(-) group (p=0.032). The TTK-, LY6K-, and IMP-3-specific CTL responses were observed after the vaccination in 45%, 63%, and 57% of patients in the 24(+) group, respectively, and the peptide-specific responses were confirmed by the pentamer assay. In the 24 (-) group, the patients showing the peptide-specific CTL response analyzed by dump assay were observed in 12% of them, indicating the cross-reactivity against the vaccinated peptides with different HLA allele. The OS in patients having all of three peptide-specific CTL responses indicated much better prognosis (OS=7.5 month) compared to that in the other patients. Conclusions: The phase II cancer vaccine therapy demonstrated the promising survival benefit as well as good immunogenicity, and, therefore, warrants further clinical studies.