Freshwater salinization has become an increasing threat to the culture of the freshwater pearl mussel Hyriopsis cumingii. To evaluate its physiological tolerance and regulatory responses to salinity stress, mussels were exposed to 0, 2, 4 and 6‰ salinity, and changes in survival, osmotic regulation, antioxidant capacity, energy metabolism, histology and transcriptomic profiles were examined. Rising salinity significantly reduced survival and disturbed osmotic homeostasis, as reflected by increased hemolymph osmolality, tissue water loss, Na+/K+-ATPase activity, and the accumulation of alanine and glutamate. Histological observations further showed progressive damage in the gill and hepatopancreas with increasing salinity. Salinity stress also induced oxidative and metabolic adjustments, including increased catalase activity, altered superoxide dismutase activity, and significant changes in key tricarboxylic acid cycle enzymes, free amino acids and free fatty acids, indicating enhanced aerobic metabolism and the preferential mobilization of amino acid and lipid substrates. Transcriptomic analysis revealed that differentially expressed genes were mainly enriched in MAPK and NF-kappa B signaling, amino acid metabolism, glycolysis/gluconeogenesis, the tricarboxylic acid cycle and fatty acid metabolism, suggesting coordinated regulation of immune defense, osmotic adjustment and energy reprogramming. These results indicate that salinity stress triggers a short-term compensatory response rather than effective high-salinity adaptation in H. cumingii. The species retained limited compensatory capacity at 2‰, whereas salinities above 4‰ exceeded its regulatory range, with 6‰ causing severe stress. This study provides a physiological and molecular basis for salinity management and stress-resistance breeding in freshwater pearl mussel culture.