Copper (Cu) contamination has become a severe problem in freshwater environments worldwide. The freshwater mussel Anodonta woodiana is used as a unique bioindicator to monitor Cu contamination in freshwater environments. However, Cu toxicity and response mechanisms in A. woodiana are still largely unknown. A sublethal acute exposure experiment (2.0 mg/L Cu exposure for 72 h) was conducted to investigate the effects of Cu bioaccumulation on ionoregulatory homeostasis, histological features, and transcriptome responses using A. woodiana gills as indicator tissue. The gill bioaccumulation capacity was up to 474. Cu bioaccumulation decreased Na+ and Mg2+ concentrations (p < 0.05) by 82% and 17%, respectively, and induced cilia loss, epithelial desquamation, and filament atrophy of the gills. Transcriptome analysis identified 3160 differentially expressed genes (DEGs), including 1870 upregulated and 1290 downregulated genes. GO enrichment analysis showed that cellular processes, metabolic processes, biological regulation, and responses to stimuli contained the most DEGs in the biological processes. KEGG pathway analysis showed that apoptosis, arginine and proline metabolism, the toll-like receptor signaling pathway, apoptosis-multiple species, histidine metabolism, beta-alanine metabolism, cytokine–cytokine receptor interaction, and the p53 signaling pathway were significantly enriched. These findings provide comprehensive evidence for exploring Cu toxicity and response mechanisms in freshwater mussels.