According to field surveys, the Red-eared Slider (Trachemys scripta elegans) can survive in brackish water although it generally lives in fresh water. In order to understand protein utilization under acute salinity stress, the changes of free amino acids in muscle and soluble protein content in blood and liver at salinity 5, 10 and 15 (that is 5‰, 10‰ and 15‰ salinity) and control group at 0 h, 24 h and 96 h were studied. As shown in Table 1, total free amino acid contents in the muscle of Red-eared Slider in three salinity groups were significantly higher than in the control group at 24 h and 96 h (P < 0.05). The contents of asparagine, histidine, alanine, glutamine and glutamic acid were rich. There was no significant difference in the contents of glutamine, glutamic acid, glycine, leucine, tryptophan, valine and methionine at different sampling time points (P > 0.05). However, the contents of asparagine, alanine, arginine, proline and aspartic acid increased with the extension of sampling time in salinity groups. Principal component analysis showed that serine, alanine, arginine, proline, tyrosine, isoleucine, phenylalanine, lysine, aspartic acid, histidine, glutamine and glutamic acid and glycine play a major role in osmotic regulation as shown in Table 2. According to analysis of two-way ANOVA, there were significant differences and interaction effect of salinity and sampling time in the content of serine, alanine, arginine, proline, tyrosine, isoleucine, phenylalanine, lysine, aspartic acid and histidine as shown in Table 3 (P < 0.05). The contents of soluble proteins in the blood of the salinity groups decreased with ambient salinity increase, and those in salinity 10 and 15 were significantly lower than the control group at 24 h and 96 h in Fig. 1 (P < 0.05). However, the content of soluble proteins in the liver in salinity 5 was significantly higher than tha of the control group at 24 h, while significantly lower than that of the control group at 96 h (Fig. 2, P < 0.05). Therefore, we conclude that the Red-eared Slider can produce free amino acids which are released into the blood to maintain osmotic balance through the metabolism of amino acids in the muscle and decomposition of soluble protein in the blood and liver. These results will provide an important reference for the study of physiological invasive mechanism in the Red-eared Slider and improve the osmoregulatory mechanism of the turtles.