Hepatitis B core antigens (HBcAg) and hepatitis B surface antigens (HBsAg) are the main structural antigens of hepatitis B virus (HBV). Both antigens are potent immunogens for experimental animals as well as in acutely infected patients. A novel formulation based on the combination of HBsAg and HBcAg has been developed as a therapeutic vaccine candidate, aimed at inducing an immune response capable of controlling the infection. An immunization schedule was conducted to evaluate the immunogenicity of this formulation after simultaneous immunization by the intranasal and parenteral routes using different schedules and doses. Humoral and cellular immune responses generated in blood and spleen were evaluated by engyme-linked immunosorbent assay (ELISA) and enzymeliked immunospot (ELISPOT) assays respectively. A first experiment evaluated two groups of mice simultaneously immunized by intranasal (IN) and subcutaneous (SC) routes, one including alum by SC route and, in the other, the formulation was injected without adjuvant. As a result, alum adjuvant did not increase the immunogenicity under the studied conditions. In fact, the group without alum induced the most potent immune response. The immune response was enhanced by combining IN and SC immunization compared to the SC route alone. In a second experiment, mice were immunized by different mucosal routes at the same time, and compared to the simultaneously (IN/SC) immunized groups. It was demonstrated that there is no improvement on the resulting immune response by using multiple routes of immunizations simultaneously; however, the increase of the antigen dose induced a superior immune response. Interestingly, the increase of antigen dose only by SC route did not favor the resulting immunogenicity. In conclusion, the use of HBsAg transgenic mice has proven useful to optimize the formulation, avoiding the unnecessary use of alum as adjuvant as well as provided information of the role of different mucosal immunization routes and antigen dose on the resulting immune response.