Previous studies have demonstrated that the therapeutic vaccine based on the enhancement of hepatitis B virus (HBV)-specific cytotoxic T lymphocyte (CTL) activity may lead to viral clearance in HBV-infected individuals. The endoplasmic reticulum (ER) chaperone Tapasin plays an important role in major histocompatibility complex (MHC) class I assembly and enhances specific MHC class I-restricted CTL activity by allowing more peptides to be translocated into the ER. Combining the specificity of hepatitis B core antigen (HBcAg) CTL epitope, the cell-penetrating property of cytoplasmic transduction peptide (CTP), and chaperone Tapasin may elicit robust specific HBV immune responses. In the present study, we confirmed the cytoplasmic localization preference of CTP-HBcAg(18-27)-Tapasin fusion protein in vitro and evaluated the effects on promoting bone marrow-derived dendritic cells (BMDCs) maturation and enhancing T cells response to generate specific CTLs. Our results showed that CTP-HBcAg(18-27)-Tapasin fusion protein could not only penetrate into the cytoplasm exactly and effectively to elevate Tapasin expression, but also increase the expression of surface molecules (CD80, CD83, CD86, and MHC-I) and secretion of cytokine (IL-12p70) of DCs. Moreover, DCs treated with the above fusion proteins increased significantly the cytokine secretion of proliferated T cells in vitro, the percentages of IFN-γ(+)CD8(+) T cells and specific CTL responses compared with control groups. In conclusion, the modification of Tapasin can enhance the presentation of targeting antigens via intracellular delivery to DCs and elicit specific CTL immune responses efficiently.