This study aimed to investigate the effects of hypoxia on Cu-induced antioxidant defense, Cu transport, and mitophagy in the liver of the large yellow croaker. Fish were exposed to hypoxia (3.0 mg L-1), Cu (120 μg L-1), and hypoxia (3.0 mg L-1) plus Cu (120 μg L-1) for 48 h. Hypoxia exposure increased antioxidant abilities to maintain cellular redox balance. Although Cu exposure alone improved antioxidant defense, Cu transport, and mitophagy, these stress responses could not completely neutralize Cu toxicity, as reflected by the elevated reactive oxygen species (ROS) and lipid peroxidation (LPO) and hepatic vacuoles. When compared with Cu stress alone, hypoxia increased Cu toxicity by inhibiting antioxidant defense, Cu transport, and mitophagy, leading to the increment of mortality, ROS, and LPO, and the deterioration of histological structure. The adverse effects of hypoxia on Cu-induced metal transport and mitophagy might be involved in metal-responsive element-binding transcription factor-1 (MTF-1) and Forkhead box O-3 (FoxO3) signaling pathways, respectively. Overall, hypoxia reduced antioxidant response, Cu transport, and mitophagy in fish exposed to Cu, which contributes to understanding the molecular mechanisms underlying negative effects of hypoxia on Cu toxicity in fish.