Background: The peptides derived from ideal cancer-testis antigens, including LY6K, CDCA1 and IMP3 (identified using genome-wide cDNA microarray analyses), were utilized in immunotherapy for head and neck squamous cell cancer (HNSCC). In this trial, we analyzed the immune response to and safety and efficacy of vaccine therapy. Methods: A total of 37 patients with advanced HNSCC were enrolled in this trial of peptide vaccine therapy, and the OS, PFS and immunological response were evaluated using enzyme-linked ImmunoSpot (ELISPOT) and pentamer assays. The peptides were subcutaneously administered weekly with IFA. The primary endpoints were evaluated based on differences between HLA-A*2402-positive (A24(+)) patients treated with peptide vaccine therapy and –negative (A24(-)) patients treated without peptide vaccine therapy among those with advanced HNSCC. Results: Our cancer vaccine therapy was well tolerated. The OS of the A24(+)vaccinated group (n = 37) was statistically significantly longer than that of the A24(-) group (n=18) (MST 4.9 vs. 3.5 month, respectively, p<0.05). One of the patients exhibited a complete response. In the A24(+) vaccinated group, the ELISPOT assay identified LY6K-, CDCA1- and IMP3-specific CTL responses in 85.7%, 64.3% and 42.9% of the patients, respectively. The patients showing LY6K- and CDCA1-specific CTL responses demonstrated a longer OS than those without CTL induction. Moreover, the patients exhibiting CTL induction for multiple peptides demonstrated better clinical responses. Conclusions: The immune response induced by this vaccine may improve the prognosis of patients with advanced HNSCC. Clinical trial information: 000008379.