To assess the role of dietary creatine on myofibre characteristics and protein synthesis in muscle, we fed grass carp (Ctenopharyngodon idellus, initial body weight: 88·47 ± 1·44 g) creatine-supplemented diets (1·84, 5·91, 8·48 and 15·44 g/kg diet) for 8 weeks. Creatine supplementation did not affect growth performance, but significantly increased creatine contents in muscle and liver. At 8·48 g/kg, creatine decreased the activities of alanine transaminase and aspartate aminotransferase in serum and improved hardness and chewiness of muscle due to shorter myofibre mean diameter, higher myofibre density and the frequencies of the diameters of classes I and III and collagen content, longer sarcomere length and upregulated mRNA levels of slow myosin heavy chains. Creatine supplementation upregulated the mRNA expressions of myogenic regulatory factors. The 8·48 g/kg creatine-supplemented diet significantly increased the contents of protein, total amino acids (AA), essential AA and free flavour AAs in muscle, the protein levels of insulin-like growth factor I, myogenic differentiation antigen and PPAR-γ coactlvator-1α in muscle and stimulated the phosphorylation of target of rapamycin (TOR) pathway in muscle. In summary, 8·48 mg/kg creatine improved fish health and skeletal muscle growth and increased hardness and protein synthesis in muscle of grass carp by affecting myofibre characteristics and the TOR signalling pathway. A second-order regression model revealed that the optimal dietary creatine supplementation of grass carp ranges between 8·48 and 12·04 g/kg.