Abstract: This study aimed to investigate the effects of dietary supplementation with arginine (Arg) on osmoregulation, antioxidant status, and immune capacity of rainbow trout Oncorhynchus mykiss before and after seawater acclimation. A total of 480 rainbow trout (initial weight of 67.04±6.93 g) were randomly divided into four groups. Four experimental diets were formulated to contain Arg level of 1.46% (A-1.46), 2.50% (A-2.50), 3.45% (A-3.45), and 4.62% (A-4.62), respectively. After five weeks of freshwater culture, each group of rainbow trout underwent seawater acclimation for 2 days and 4 days, designated as SA-2 and SA-4, respectively. SA-2 entails a gradual increase in salinity from 0 to 15 on the first day, followed by reaching the local seawater salinity of 27 on the second day. SA-4 followed a similar increase as SA-2 on the first day, then increased by 4 units per day until reaching as a salinity of 27. Samples were collected at the conclusion of freshwater culture and at 1d, 7d, and 21d after seawater transfer to evaluate osmoregulation, antioxidant, and immune parameters. Results showed that at the end of freshwater culture, the A-4.62 group had significantly lower final body weight and percent weight gain, indicating that excessive dietary Arg hinder their growth. Under both acclimation conditions, serum K⁺ levels in the A-3.45 and A-4.62 groups initially increased and then normalized over time, suggesting effective ion regulation in these Arg groups. After 21d after seawater transfer, the liver total antioxidant capacity (T-AOC) of all groups significantly surpassed their respective freshwater values, with the A-3.45 group displaying the lowest integrated biological response (IBR), indicating reduced stress in this group. Compared to the SA-2 method, rainbow trout in all SA-4 groups maintained more stable osmotic pressure, suggesting SA-4’s superiority in maintaining osmotic balance. Additionally, under SA-2, the A-3.45 group excreted excess Na⁺ to better adapt in seawater; serum lysozyme (LZM) levels notably rose over time in the A-3.45 group, indicating an enhanced immune response in these rainbow trout after seawater transfer under SA-2. Under SA-2, after 21d of seawater transfer, the A-3.45 group exhibited reduced gill NKA activity to decrease Cl^– influx, thereby better maintaining ion balance. In summary, under both acclimation conditions, rainbow trout demonstrated enhanced growth and adaptability when dietary Arg content was 3.45%.