HlyIIR is a negative transcriptional regulator of the hemolysin II gene from Bacillus cereus. A disordered region (amino acid residues 170-185) localized within the C-terminal domain near the dimerization interface was found in the recently determined HlyIIR X-ray structure. To clarify the effect of this region on HlyIIR properties and potential improvement of the diffraction quality of its crystals, we constructed an HlyIIR mutant with a single alanine residue substituting for the overall disordered region. According to biochemical analysis, the mutant protein still formed a dimer but lost its DNA-binding activity. Its crystals displayed better diffraction quality as compared with the native protein. The mutant structure was determined by X-ray analysis with a resolution of 2.1 Å. However, the mutant protein formed an alternative dimer differing from the wildtype dimer, as its subunits were rotated by 160°. The conformation of individual subunits also partially changed. Because this considerable remodeling in the mutant protein structure resulted from the conformational changes in the segment Pro161-Ser169, we concluded that this segment was important for maintaining the native HlyIIR structure.