Over 160 million cases of shigellosis occur annually worldwide, with the two most prevalent species being Shigella flexneri and S. sonnei. Protective immunity against Shigella infection is primarily directed at the lipopolysaccharide (LPS) of the homologous serotype, so it may be necessary to combine monovalent vaccines for multiple Shigella serotypes to construct a multivalent vaccine against predominant serotypes. Recently, we described a subcellular vaccine isolated from virulent S. flexneri, consisting of proteins (including the invasins IpaB and IpaC) and LPS, that protected mice and guinea pigs from homologous challenge. In the present study, a bivalent Invaplex vaccine consisting of S. flexneri 2a and S. sonnei Invaplex was used to intranasally immunize mice and guinea pigs to determine the bivalent vaccine's immunogenicity and protective capacity against challenge with either strain. Mice and guinea pigs immunized with the bivalent S. flexneri 2a/S. sonnei Invaplex vaccine produced serum IgA and IgG antibodies to S. flexneri LPS, S. sonnei LPS, the homologous Invaplex and the water extract antigens (invasins) as determined by ELISA. The immune responses in animals immunized with the bivalent vaccine were similar to responses in animals immunized with the monovalent Invaplex vaccines. Mice and guinea pigs immunized with the bivalent vaccine were protected from a lethal lung challenge (mice, P<0.001) or severe keratoconjunctivitis (guinea pigs, P≤0.002) after challenge with either S. flexneri 2a or S. sonnei. Animals immunized with monovalent Invaplex vaccines were protected (P<0.001) against the homologous agent at levels comparable to the bivalent vaccine. After challenge, immunized animals demonstrated boosts in antibody titers to LPS, water extract antigens and Invaplex. These studies indicate that the subcellular Invaplex vaccine will be readily adaptable to a multivalent vaccine approach for shigellosis.