The large yellow croaker Larimichthys crocea is an important marine fish species. The output of this fish is the highest among the sea cage-cultured fishes in recent years in China, however, this species is susceptible to Cryptocaryon irritans, which causes a high death rate. Although CuSO₄ is currently the best treatment for C. irritans, it can lead to Cu pollution in aquaculture water bodies. At the same time, low temperatures have resulted in mass mortality during overwintering. To evaluate the effects of Cu acclimation on oxidative damage and gene expression in the liver of large yellow croaker under cold stress, fish (74.6 ± 4.2 g) were pre-exposed to 0 and 10 μg Cu/L for 14 d and subsequently subjected to cold stress (8 ℃) for 24 h. The results indicated that cold stress enhanced reactive oxygen species (ROS) and lipid peroxidation (LPO), indicating that cold stress had a negative effect on fish. Although Cu acclimation had no effect on ROS and LPO, Cu acclimation with cold stress increased ROS and LPO compared with cold stress alone, suggesting that Cu acclimation exacerbated cold stress-induced oxidative damage. A total of 2288, 1425, and 1382 differentially expressed genes (DEGs) were obtained from the Cu acclimation group vs. control, cold stress group vs. control, and Cu acclimation with cold stress group vs. cold stress group, respectively. The results of GO functional enrichment and KEGG pathway enrichment showed that DEGs were significantly enriched in the PPAR signaling pathway, fatty acid elongation, unsaturated fatty acid biosynthesis, glycolysis/gluconeogenesis, oxidative phosphorylation, glutathione metabolism, endoplasmic reticulum protein processing, FoxO signaling pathway, regulation of autophagy, MAPK signaling pathway, and apoptosis, highlighting the adaptive mechanism of fish in response to Cu and cold stresses involved in fatty acid metabolism, energy metabolism, antioxidant, endoplasmic reticulum stress, autophagy, and apoptosis. Cluster analysis showed that cold stress upregulated the majority of gene expression related to unsaturated fatty acid synthesis, endoplasmic reticulum stress, autophagy, and apoptosis. However, Cu acclimation had an antagonistic effect on the regulation of these genes under cold stress, indicating that Cu acclimation reduced the cold stress tolerance of the large yellow croaker by inhibiting unsaturated fatty acid synthesis, endoplasmic reticulum stress, autophagy, and apoptosis. These results provide a scientific basis for the study of the effects of Cu contamination on cold stress tolerance in the large yellow croaker and the underlying molecular mechanism.