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The diet formulation for trout has changed dramatically over the last decade due to changes in the ingredient markets and advances in feed processing technology. The protein requirements of Oncorhynchus mykiss were established at the end of the last century, and it is unclear whether these requirements are applicable to modern dietary formulations. Therefore, an eight-week feeding trial was performed to measure the protein requirements of O. mykiss by evaluating growth, body composition, antioxidation property, innate immune response and liver morphology. The five experimental diets were prepared to contain the same levels of crude lipid (120 g/kg) and graded levels of crude protein (356.3, 383.9, 411.5, 439.2 and 466.8 g/kg). The results suggested that the growth, feed utilization and whole-body crude protein levels were significantly increased when fish were fed diets containing 439.2 and 466.8 g/kg crude protein. Meanwhile, low dietary protein levels (356.3 and 383.9 g/kg) significantly down-regulated the mRNA levels of insulin-like growth factor I, catalase, glutathione peroxidase, superoxide dismutase, complement 3 and lysozyme, and also up-regulated the insulin-like growth factor binding protein 1 as well as proinflammatory cytokine expression in the liver, including interleukin 1β, interleukin 8 and tumor necrosis factor-α. Moreover, low dietary protein levels (356.3 and 383.9 g/kg) damaged liver structure, suppressed total antioxidative capacity and increased the malondialdehyde content in liver. In conclusion, high dietary protein (439.2 and 466.8 g/kg) promoted fish growth, while low dietary protein (356.3 and 383.9 g/kg) damaged liver structure, induced oxidative stress and inflammatory responses and weakened non-specific immunity. The protein requirement of O. mykiss reared in the convection-water cages is no less than 439.2 g/kg for optimal growth, antioxidant and immune properties.