Glypican-3 (GPC3), which is a carcinoembryonic antigen, is overexpressed in human hepatocellular carcinoma (HCC). Previously, we performed a phase I clinical trial of GPC3‑derived peptide vaccination in patients with advanced HCC, and reported that GPC3 peptide vaccination is safe and has clinical efficacy. Moreover, we proposed that a peptide‑specific CTL response is a predictive marker of overall survival in patients with HCC who receive peptide vaccination. In this study, we established GPC3‑derived peptide-specific CTL clones from the PBMCs of an HLA-A*02:07-positive patient with HCC who was vaccinated with an HLA-A2-restricted GPC3 peptide vaccine and showed a clinical response in the phase I clinical trial. Established CTL clones were analyzed using the IFN-γ ELISPOT assay and a cytotoxicity assay. GPC3 peptide-specific CTL clones were established successfully from the PBMCs of the patient. One CTL clone showed cytotoxicity against cancer cell lines that expressed endogenously the GPC3 peptide. The results suggest that CTLs have high avidity, and that natural antigen-specific killing activity against tumor cells can be induced in a patient with HCC who shows a clinical response to vaccination with the GPC3144-152 peptide.