Vaccines against Shiga toxin (Stx)-producing Escherichia coli (STEC) have not yet been developed. Two immunologically distinct serotypes of Stx (Stx1 and Stx2) are the main virulence factors of STEC. Thus, blocking their B subunits (StxB) from binding to the cell surface receptor globotriaosylceramide (Gb3) efficiently prevents the action of these toxins. We expressed Stx1B and Stx2B in E. coli inclusion bodies and reassembled them into pentamers by a stepwise dialysis. Stx1B pentamer fully protected mice against Stx1 challenge, but Stx2B pentamer failed to protect mice against Stx2 challenge. To explain those observations, we proposed that the pentamer of Stx2B readily dissociates into its constituent monomers, especially under in vivo conditions, thus being unable to induce pentamer-specific immunity. To increase pentamer stability, we fused the B subunit to a pentameric coiled-coil domain of the cartilage oligomeric matrix protein (COMP). This “five-to-five” fusion hybrid molecule (Stx2B–COMP) was shown to be protective against Stx2 challenge, demonstrating that the Stx2B subunit when leashed and bundled by a rigid pentameric coiled-coil domain mount a pentamer-specific immune response and efficiently neutralize the toxin both in vitro and in vivo. Our data strongly suggest that the Stx2B subunit moiety fluctuates between a pentameric and monomeric state within the fusion protein, which may increase the likelihood of the immune system recognizing the pentameric conformation for toxin neutralization.