Live attenuated Listeria monocytogenes (LM) is a promising bacterial vector able to induce a T-cell response to tumor-associated antigens and demonstrates great potential for use in vaccine development. A novel recombinant LM-based vaccine (Lmdd (LM ΔdalΔdat)-MPFG (multiple peptide fusing genes)) was developed with the ability to express and secrete hepatocellular carcinoma (HCC)-related tumor-associated antigens fragments due to the insertion of hepatitis B virus (HBV)-X protein (HBx)-derived epitopes HBx(52-60) and HBx(140-148), the universal T-helper epitope, alpha-fetoprotein (AFP) epitope AFP(158-166), and melanoma antigen gene (MAGE)-3(271-279) into the HBV core protein. Following immunization with the Lmdd-MPFG vaccine, macrophages exhibited uptake of the bacteria; the vaccine was then nearly cleared 3 days after the first administration. It disappeared even more quickly following subsequent vaccinations. However, recombinant Lmdd-MPFG allowed for the full development of an antitumor response towards the human leukocyte antigen (HLA)-A0201 epitopes of MPFG. Each epitope stimulated an augmented T-cell proliferation and enhanced the supernatant level of interferon (IFN)-γ in vitro. In addition, IFN-γ-producing CD8(+) T cells as well as in vivo cytolytic activity were significantly increased in HLA-A2 transgenic mice. Additionally, the Lmdd-MPFG developed a strong antitumor response, as indicated by the significant resistance of immunized mice to MPFG-positive Hepa1-6 cell challenge in both a prophylactic and therapeutic setting. Tumor regression was accompanied by an enhanced cytotoxic T lymphocyte response and a decrease of regulatory T cells in the tumor. Collectively, these results suggest that utilizing attenuated LM as a vaccine vector, able to carry the MPFG gene, presents a potentially feasible strategy for prevention of HCC.