Recombinant fusion proteins containing domain III of the dengue envelope protein fused to the P64k protein from Neisseria meningitidis and domain III of dengue 2 fused to capsid protein of this serotype were immunogenic and conferred protection in mice against lethal challenge, as previously reported. Combining the domain III-P64k recombinant proteins of dengue 1, 3 and 4 with the domain III-capsid protein from dengue 2, we obtained a novel tetravalent formulation containing different antigens. Here, the IgG and neutralizing antibody response, the cellular immune response and the protective capacity against the lethal challenge in mice immunized with this tetravalent formulation were evaluated. The neutralizing antibody response obtained against D1, D2 and D3 together with the high levels of IFNγ secretion induced after stimulation with the four dengue serotypes support the strategy of a new tetravalent formulation containing the domain III of envelope protein fused to the capsid protein of each dengue serotype.