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Keap1 is deemed as a suppressor of Nrf2 in cytoplasm by sequestrating Nrf2 to proteolysis as an adapter of the Cul3-Rbx1 E3 ubiquitin ligase complex. In the study, it was proposed that post-translational modification might affect the interaction between Nrf2 and Keap1, and the profiles of the phosphorylation of amino acid residues of Keap1 and its effects on the binding of Keap1 to Nrf2 was investigated. A mass spectrometry analysis revealed that S53 and S293 were phosphorylated upon an oxidative stress. Using Keap1 proteins with amino acid residues mutated to glutamate to simulate the introduction of a negative charge by phosphorylation, it was found that a potential phosphorylation of S53 affected Keap1-Nrf2 binding in the pull-down assay, and induced nuclear translocation of Nrf2 in the electrophoretic mobility shift assay. Sequence homology analysis showed that S53 was highly conserved. Structural modeling around BTB domain of wild type and S53E-mutant Keap1 showed that the negative charge introduced by S53E mutation generates a salt bridge between E53 and ionized guanidine group of Arg50. Real-time qRT-PCR for transcription levels of antioxidant genes that are modulated by Nrf2 further proved the effects of the potential phosphorylation of S53 under an oxidative stress condition. In summary, S53 is a potential phosphorylation site of Keap1, and the phosphorylation could enhance the antioxidative capacity of cells in response to an oxidative stress.