Nuclear factor-kappa B (NF-kappaB) is an important transcription factor, involved in many immune and inflammatory responses. It is critical in HIV gene expression as it has kappa B binding sites in the HIV-1 long-terminal repeat. Hence, targeting NF-kappaB to prevent its DNA binding holds a significant therapeutic potential. In this context, we report Evans Blue as a novel inhibitor of NF-kappaB-DNA binding. Evans Blue was found to be inhibiting DNA binding of NF-kappaB at a low concentration of 100 microM. Further, molecular modeling studies using docking and generation of electrostatic potential maps predicted a possible binding mode of EB to the DNA binding region of NF-kappaB, consistent with the experimental activity.