Six fluorescent fusion protein plasmids were constructed, and the location and interaction of p53 and IκBα (IκBαM, IκBα243N and IκBα244C) in living cells were investigated with cyan (CFP) and red fluorescent protein(DsRed) fusion proteins by fluorescence resonance energy transfer. p53 interacts with the non-ankyrin C terminus PEST domain of IκBα, and the N terminus in p53 plays an important role in the formation of p53·IκBα complex. The change of DsRed/CFP in response to UV-mediated DNA damage was observed in real time and the balance between p53·IκBα complex formation and dissociation was discussed. In addition, the shuttling of p53 and IκBα between cytoplasm and nucleus under LMB stimulation suggested that the relationship among NF-κB, p53, IκBα, Mdm2, and WOX1 is complicated and remains to be determined. IκBα has been an important cross-talk protein between p53 and NF-κB, and this might expand the understanding of these two signaling pathways considerably.