Keap1-Nrf2 signaling pathway is one of the most important antioxidant signaling pathways, and its abnormal activation is related to cancer metastasis and drug resistance. Many studies have shown Keap1 and Nrf2 mutations are closely associated with cancer occurrence. However, few studies focus on Keap1-Nrf2 binding characteristics of cancer-associated mutations. The study investigated the molecular mechanism between Keap1/Nrf2 mutations and cancer.We have determined the crystal structure of the Keap1-Kelch domain with Nrf2 25-mer peptide. What's more, we clarified the molecular effects of Nrf2Thr80 and Nrf2Pro85 on the binding of Keap1 by the method isothermal titration calorimetry (ITC), differential scanning fluorimetry (DSF) and electrophoretic mobility shift assay (EMSA). Especially, we confirmed the effect of Thr80 and Pro85 mutations on Keap1/Nrf2 signaling pathway in HEK293T cells by RT-PCR and western blot (WB). Finally, we verified the effect of six cancer-related high-frequency somatic mutations Keap1G364C, Keap1D422N, Keap1R470C, Keap1G480W, Keap1E493Q and Keap1R601L on binding with Nrf2 through ITC experiments.Nrf2Thr80 and Nrf2Pro85 play a vital role in the Keap1-Nrf2 interaction. Mutant or modification at position Thr80 will disrupt the interaction. Especially, Nrf2Thr80 and Nrf2Pro85 mutations activate the expression of cytoprotective genes in HEK293T cells. As for Keap1, except G364C, the binding affinity of other cancer-related mutants to Nrf2 hardly changed, which means that Keap1 mutants can activate Nrf2 without disrupting the binding to Nrf2.The study provides new insight into Keap1/Nrf2 signaling pathway and cancer.Copyright © 2021 Elsevier Inc. All rights reserved.