In tilapia aquaculture, the transfer of the fish to hyperosmotic seawater (SW) is one of the essential steps for diminishing undesirable flavor in muscle. In this study, Mozambique tilapia (Oreochromis mossambicus) was utilized to investigate glutamate/glutamine-related metabolism in muscle under various salinity treatments. Transcript expressions of glutamate-ammonia ligase (GLUL) and glutamate dehydrogenase (GLUD) in muscle were found to be significantly increased in 10 psu SW. In addition, the contents of glutamine and other amino acids in muscle were found to be increased in both 10 and 20 psu SW. In contrast, the ammonia contents in muscle were not affected by different SW conditions. These results indicated that the glutamine accumulated in muscle may be generated by the coupling of glutamate with ammonia following 10 psu SW treatment. These physiological strategy utilized by aquaculture tilapia muscle under hyperosmotic stress would cause the accumulation of amino acids and further induce the “umami” flavor of human taste.