Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor for cytoprotective gene activation in the oxidative stress response. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses the transcriptional activity of Nrf2. During oxidative stress, Nrf2 is released from Keap1 and is translocated into the nucleus, where it interacts with the small Maf protein to initiate gene transcription. Prothymosin α (ProTα), an intrinsically disordered protein, also interacts with the Kelch domain of Keap1 and mediates the import of Keap1 into the nucleus to inhibit Nrf2 activity. To gain a molecular basis understanding of the oxidative stress response mechanism, we have characterized the interaction between ProTα and the Kelch domain of Keap1 by using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, peptide array analysis, site-directed mutagenesis, and molecular dynamic simulations. The results of nuclear magnetic resonance chemical shift mapping, amide hydrogen exchange, and spin relaxation measurements revealed that ProTα retains a high level of flexibility, even in the bound state with Kelch. This finding is in agreement with the observations from the molecular dynamic simulations of the ProTα-Kelch complex. Mutational analysis of ProTα, guided by peptide array data and isothermal titration calorimetry, further pinpointed that the region (38)NANEENGE(45) of ProTα is crucial for the interaction with the Kelch domain, while the flanking residues play relatively minor roles in the affinity of binding.