Abstract: In order to investigate the effects of acute ammonia nitrogen stress on the antioxidant and immune function of rainbow trout (Oncorhynchus mykiss) in an alkaline environment, fish were exposed to three different concentrations of ammonia nitrogen (0.86, 1.73, and 2.59 mg/L) for up to 96h. We measured the activities of antioxidant enzymes (CAT, T-AOC, T-SOD, and MDA) and immune enzymes (ACP, AKP, GOT, and GPT) in the liver and serum, as well as the expression levels of related genes in the liver. The results showed that antioxidant enzyme activity in the liver increased from 0 to 6h and then decreased, while T-AOC activity in the serum exhibited a trend of decreasing followed by increasing under different concentrations of ammonia nitrogen stress. At 48h, MDA content was the lowest and T-SOD activity was the highest with the high concentration group in the liver. In the different concentration groups, immune enzyme activity increased from 0 to 6h and then gradually decreased in both liver and serum. At 96h, GPT activity was significantly higher than that of the control group (P<0.05) in both liver and serum. In the low, medium, and high concentration groups, the expression levels of antioxidant related genes (cu/zn-sod, mn-sod, and cat) were higher than those of the control group at 6h in the liver. At 12h, the expression of the immune related gene hsp90 was higher than that of the control group, whereas hsp70 showed the opposite trend. These results indicated that antioxidant and immune enzyme activities were induced, and the expression of related gene was upregulated at the early stage of stress. With prolonged stress, antioxidant and immune functions were inhibited, as evidenced by the downregulation of enzyme activities and gene expressions. This study provides fundamental information for further investigation on the stress mechanisms of antioxidant and immune systems in rainbow trout under ammonia nitrogen stress.