HlyIIR is a negative transcriptional regulator of hemolysin II gene from B. cereus. It binds to a long DNA perfect inverted repeat (44bp) located upstream the hlyII gene. Here we show that HlyIIR is dimeric in solution and in bacterial cells. No protein-protein interactions between dimers and no significant modification of target DNA conformation upon complex formation were observed. Two HlyIIR dimers were found to bind to native operator independently with Kd level in the nanomolar range. The minimal HlyIIR binding site was identified as a half of the long DNA perfect inverted repeat.