Hexavalent chromium [Cr (VI)] and its compounds (eg chromates), which are extensively used in numerous industrial processes including leather tanning and steel manufacturing, are considered as priority pollutants. There is growing evidence supporting that Cr (VI) could be a human carcinogen that induces primary liver cancer after oral exposure, and this sheds light on the importance of the investigation of Cr (VI)-induced hepatotoxicity. Although it is known that mitochondria are major targets for heavy metals, the mechanisms of electron transfer chain (ETC) dysfunction involved in Cr (VI)-induced cytotoxicity are unclear. In the present study, by using mitochondrial respiratory chain complex (MRCC) I inhibitor rotenone (ROT) and its substrates glutamate/malate (Glu/Mal), MRCC III inhibitor antimycin A (AA) and its substrate coenzyme Q (CoQ), and the antioxidant Vitamin C (Vit C), we aimed to elucidate the role of mitochondrial ETC dysfunction in Cr (VI)-induced cytotoxicity. We found that Cr (VI) targeted and inhibited MRCC I and III to induce ETC dysfunction, which played an important role in Cr (VI)-induced cytotoxicity. Our current data provides novel important insight into the mechanisms of mitochondrial ETC dysfunction in Cr (VI)-induced cytotoxicity in the hepatocytes, and we will be poised to develop new methods in the prevention and treatment of liver diseases involving mitochondrial ETC dysfunction for the occupational exposure population.