The molecular basis of gene regulation by Nuclear Factor-κB (NF-κB) transcription factors and their coregulators is not well understood. This family of transcription factors controls a number of essential subcellular processes. Human Pirin, a nonheme iron (Fe) binding protein, has been shown to modulate the binding affinity between p65 homodimeric NF-κB and κB DNA. However, the allosteric effect of the active Fe(III) form of Pirin on the DNA has not been established. Here, we use multiple microsecond-long molecular dynamics simulations to explore the conformational dynamics of the free DNA, the p65-DNA complex, and the Pirin-p65-DNA supramolecular complex. We show that only the Fe(III) form of Pirin enhances the affinity between p65 and the DNA in the Pirin-p65-DNA supramolecular complex, in agreement with experiments. Additionally, the results provide atomistic details of the effect of the active Fe(III) form of Pirin on the DNA upon binding to the p65-DNA complex. In general, unlike the Fe(II) form of Pirin, binding of the Fe(III) form of Pirin to the p65-DNA complex significantly alters both the conformational dynamics of the DNA and the interactions between p65 and the DNA. The results provide atomic level understanding of the modulation of the DNA as a result of a redox-specific Fe(II)/Fe(III) coregulation of NF-κB by Pirin, knowledge that is necessary to fully understand normal and aberrant subcellular processes and the role of a subtle single electron redox process in gene regulation.