The aim of this study was to evaluate the nutritional value of cottonseed protein concentrate (CPC) as a single dietary protein source and the optimal protein level for grass carp (Ctenopharyngodon idellus). An 8-week feeding trial was conducted by feeding juvenile grass carp (initial body weight: 4.68 ± 0.01 g) with six experimental diets containing graded levels of protein provided by CPC. The results showed that the optimal CPC level (CPC4) improved the growth performance and health status of grass carp. The optimal dietary protein level was estimated to be 38.61 and 38.66% based on specific growth rate (SGR) and feed efficiency (FE), respectively. The CPC4 group significantly increased the total antioxidant capacity (T-AOC) content and glutathione peroxidase (GSH-Px) activity in the hepatopancreas (p < 0.05). In addition, the CPC4 group increased the muscle T-AOC and glutathione (GSH) content and improved muscle hardness, and the gene expression of MRFs, fgf6a, myhc-7, myhc-1, myhc-4, igf-II, and tor was upregulated while mstn gene expression was downregulated (p < 0.05). Correlation analysis revealed that the optimal dietary CPC level promoted grass carp growth, health, and flesh quality by regulating the relative abundance of intestinal microbes. Furthermore, CPC6 upregulated the ko00480 (Glutathione metabolism) and ko00620 (Pyruvate metabolism) pathways compared to CPC1 (p < 0.05), possibly indicating that low dietary CPC levels adversely affected amino acid metabolism in the intestinal microbiota of grass carp, while a high level of CPC will meet the metabolic needs of the body by increasing the utilization of energy.