invasion plasmid antigen B (IpaB) plays an important role in causing shigellosis. While IpaB's protein structure, contribution to disease mechanism, and protective immunity against infection have been well studied, the significance of individual antigenic domains, especially at the N terminus, has not been systematically characterized. In an attempt to identify IpaB protein functional epitopes and to construct an optimized polyvalent multiepitope fusion antigen (MEFA) immunogen for development of a protein-based cross protective vaccine, in this study, we identified immunodominant B-cell epitopes from the IpaB N terminus, fused each epitope to carrier protein CsaB (the major subunit of enterotoxigenic Escherichia coli CS4 adhesin) for epitope fusion proteins, immunized mice with each epitope fusion protein, examined IpaB-specific antibody responses, and assessed antibody functional activity against bacterial invasion. A total of 10 B-cell continuous epitopes were identified from IpaB N terminus, and after being fused to carrier protein CsaB, each epitope induced anti-IpaB IgG responses in the intramuscularly immunized mice. While antibody invasion inhibition assays demonstrated that antibodies derived from each identified epitope were functional, epitopes 1 (LAKILASTELGDNTIQAA), 2 (HSTSNILIPELKAPKSL), and 4 (QARQQKNLEFSDKI) induced antibodies to inhibit Shigella sonnei and Shigella flexneri invasion at levels similar to those of recombinant IpaB protein, suggesting that these three IpaB epitopes can be used potentially as IpaB-representing antigens to induce protective anti-IpaB antibodies and for construction of an epitope-based polyvalent MEFA protein immunogen for vaccine development. Currently, there are no effective measures for control or prevention of infection, the most common cause of diarrhea in children 3 to 5 years of age in developing countries. Challenges in developing vaccines include virulence heterogeneity among species and serotypes. To overcome virulence heterogeneity challenge and to develop a protein-based multivalent vaccine, we targeted a panel of virulence factors, including invasion plasmid antigens, identified functional antigenic domains or epitopes as representative antigens, and applied the novel epitope- and structure-based vaccinology platform multiepitope fusion antigen (MEFA) to integrate functional antigenic domains or epitopes into a backbone immunogen to produce a polyvalent immunogen for cross protective antibodies. Identification of functional IpaB epitopes from this study enhances our understanding of IpaB immunogenicity and allows us to directly utilize IpaB epitopes for construction of a cross protective polyvalent immunogen and to accelerate development of a protein-based vaccine.