Benzo[α]pyrene (Bap) is recognized as a ubiquitous environmental pollutant among the polycyclic aromatic hydrocarbons (PAHs) class. Previous studies have shown that the hepatotoxicity of Bap is mainly caused by its metabolites, although it remains unclear whether Bap itself induces such damage. This study integrated metabolomics and chemical proteomics approaches to comprehensively identify the potential target proteins affected by Bap in liver cells. The results from the metabolomics showed that the significant changed metabolites were related with cellular redox homeostasis. CEllular Thermal Shift Assay (CETSA) showed that Bap induced protein thermal displacement of superoxide dismutase 3 (SOD3) and glutathione peroxidase 4 (GPX4), which are closely related to oxidative homeostasis. Further validation through in vitro CETSA and drug affinity response target stability (DARTS) revealed that Bap directly affected the stability of SOD3 and GPX4 proteins. The binding affinities of Bap to the potential target proteins were further evaluated using molecular docking, while the isothermal titration calorimetry (ITC) interaction measurements indicated nanomolar-level Kd values. Importantly, we found that Bap weakened the antioxidant capacity by destroying the activities of SOD3 and GPX4, which provided a new understanding of the mechanism of hepatotoxicity induced by Bap. Moreover, our provided workflow integrating metabolomics and label-free chemical proteomics, can be regarded as a practical way to identify the targets and inter-mechanisms for the various environmental compounds.Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.