The efficacy of a potential therapeutic vaccine against chronic hepatitis B virus (HBV) infection depends on the development of strong and multi-specific T cell responses. The potency of CD8+ cytotoxic T lymphocyte (CTL) responses toward HBV core antigen (HBcAg) has been shown to be critical for the outcomes of HBV chronic infection. In this study we have identified a previously undescribed HLA-A*0201-restricted HBcAg-specific CTL epitope (HBcAg₆₄₋₇₂, C₆₄₋₇₂, ELMTLATWV). T2 binding assay showed that C₆₄₋₇₂ had high affinity to HLA-A*0201 molecule. Functionally, the peptide C₆₄₋₇₂ could induce peptide-specific CTLs both in vivo (HLA-A2.1/K(b) transgenic mice) and in vitro (PBLs of healthy HLA-A2.1+ donors), as demonstrated by interferon-γ (IFN-γ) secretion upon stimulation with C₆₄₋₇₂-pulsed T2 cells or autologous human dendritic cells (DCs) respectively. HLA-A*0201-C₆₄₋₇₂ tetramer staining revealed the presence of a significant population of C₆₄₋₇₂-specific CTLs in C₆₄₋₇₂-stimulated CD8+ T cells. Furthermore, the peptide-specific cytotoxic reactivity and the production of perforin and granzyme B of CTLs also increased after stimulation with C₆₄₋₇₂-pulsed autologous DCs. These results indicate that the newly identified epitope C₆₄₋₇₂ has potential to be used in the development of immunotherapeutic approaches to HBV infection.