The aim of the present study was to evaluate the effects of Cu pre-exposure on antioxidant defense and energy metabolism in the liver of the large yellow croaker exposed to severe hypoxia. Fish were pre-acclimated to 0 and 30 μg Cu L−1 for 96 h, and subsequently exposed to 7.0 and 1.5 mg DO L−1 for another 24 h. Hypoxic stress alone increased reactive oxygen species and hepatic vacuoles. When compared to hypoxic stress alone, hypoxic stress plus Cu pre-exposure increased mortality and ROS production, and worsened histological structure by inhibiting antioxidant defense and aerobic metabolism, and enhancing anaerobic metabolism, suggesting Cu pre-acclimation aggravated hypoxia-induced oxidative damage. NFE2-related nuclear factor 2 and hypoxia-inducible factor-1α might participate in the transcriptional regulation of genes related to antioxidant response and energy metabolism, respectively. In conclusion, Cu pre-acclimation had a synergistic effect on antioxidant response and energy metabolism in fish under severe hypoxia, which contributes to understanding the molecular mechanisms underlying negative effects of Cu pre-acclimation against hypoxic damage in fish. Unlabelled Image • Hypoxia induced ROS and increased hepatic vacuoles. • Cu pre-exposure had a synergistic effect on hypoxia-induced oxidative damage. • Nrf2 mRNA level was paralleled with antioxidant gene expressions. • HIF-1α mRNA level was correlated with gene expressions of energy metabolism. [ABSTRACT FROM AUTHOR]