Objective: Hemolysin II (HlyII) is a key pathogenic factor of Bacillus cereus. This pore-forming toxin has a β-barrel spatial structure and possesses a C-terminal extension of 94 amino acid residues, designated the C-terminal domain of HlyII (HlyIICTD). In this work, site-directed mutagenesis was performed on amino acid residues located on the surface of the HlyIICTD protein globule. It was demonstrated that the C-terminal domain can exist simultaneously in several structural isoforms. The transition of the 3D HlyIICTD structure into a stable form within the water-soluble full-length toxin monomer was observed. Methods: Recombinant proteins and their mutant forms were obtained using the Escherichia coli BL21(DE3) producing strain. Their interaction with monoclonal antibodies HlyIIC-16 and HlyIIC-23 was studied by ELISA. To define the epitopes of HlyIIC-16 and HlyIIC-23, phage display, site-directed mutagenesis, gene cloning of individual parts of the HlyIICTD molecule, and 3D modeling of HlyIICTD fused to SlyD using the AlphaFold program were employed. Results and Discussion: Monoclonal antibodies against HlyIICTD interacted more effectively with intact HlyIICTD than with the full-size toxin or the chimeric protein HlyIICTD fused with SlyD. The HlyIIC-16 and HlyIIC-23 antibodies effectively inhibited each other's interaction with immobilized HlyIICTD in ELISA, indicating partial or complete overlap of their epitopes. The HlyIIC-16 and HlyIIC-23 epitopes are localized within the Gly341–Gly364 region of the HlyII protein. Conclusions: The multiplicity of structural isoforms of the C-terminal domain is disrupted when it is incorporated into the water-soluble full-size toxin monomer or into a recombinant protein containing the chaperone SlyD.