This study aimed to re-evaluate the effect of excessive arginine supplementation on growth and feed efficiency of rainbow trout, and to assess the effect of dietary arginine supplementation on hepatic amino acid composition, expression of arginase Ⅱ (ARG 2), inducible nitric oxide synthase (iNOS), and heat shock protein 70 (HSP70) genes in the intestine, and plasma arginine, ornithine, citrulline and urea levels at 0, 6, 12 and 18 hr-postprandial. Rainbow trout (body weight: 60.5–65 g) were fed diets containing 1.47 (control [CTRL]), 3.89 (3.89A) and 5.64% (5.64A) arginine for nine weeks. Higher muscle protein content was observed in 3.89A than CTRL (p < 0.05). In postprandial study, plasma arginine of 5.64A kept increased until 12 hr-postprandial and reached identical value (around 150 µg/ml) until 18 hr-postprandial in 5.64A. Significant increase of plasma arginine level was only observed in arginine supplemented group. Meanwhile, plasma citrulline level in CTRL was significantly higher than in 5.64A at 18 hr-postprandial. A significantly higher hepatic citrulline level was also observed in CTRL than in 5.64A (p < 0.05). Significantly higher plasma and hepatic free ornithine level was observed in 5.64A than CTRL while significantly higher expression of intestinal ARG2 and HSP70 was found in arginine supplemented groups. These results suggest that citrulline availability seems to be stimulated by arginine deficiency in the CTRL and the presence of arginase in the intestine to regulate excess dietary arginine.