N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine Quinone (6PPDQ) not only causes acute mortality in salmon but also induces toxicities in other living organisms. The electrophilic quinone moiety in the 6PPDQ molecular structure can participate in binding to the cysteine residues that ubiquitously exist in protein nucleophiles, which are responsible for its toxicities. Out of the 82 6PPDQ-bonded proteins found in the human model cell line A549 on the sulfhydryl-reactive proteomics platform, which enables the precise identification of covalent binding protein targets of a pollutant, we discovered three 6PPDQ-bonded mitochondrial proteins─NDUS6, COX5B, and ATP5PB─that are involved in mitochondrial dysfunction for the first time. They impede the function of mitochondria, as witnessed by the decreased enzymatic activities of mitochondrial respiratory chain Complexes I (27.63%) and IV (23.11%), the decreased cellular ATP content (19.94%), and the reduced mitochondrial membrane potential (3.2-fold), as well as the elevated mitochondrial ROS level (2.2-fold) under the environmentally relevant 8.9 μg/L 6PPDQ exposure compared to the controls. Our findings provide experimental evidence for elucidating 6PPDQ's toxicities at the molecular level, and the knowledge learned will enhance the public's awareness of the adverse impacts of environmental pollution on health.