Dietary fat is an essential nutrient for fish growth. The appropriate sources and levels of fat in diets can not only meet the normal energy metabolism and physiological needs of culture fish, but also regulate its growth, immune, and reproductive performance. At present, a large number of studies on the utilization of fat sources in diets indicate that the demand for different fat sources in fish is essentially the demand for different types of fatty acids. Fatty acids are diverse and widely distributed in nature, and the absorption and utilization of dietary fatty acids by fish often vary depending on the length of the carbon chain. Previous studies have shown that the effects of fatty acids with different carbon chain lengths on fish are mostly concentrated on growth and development, gut microbiota, and lipid metabolism. However, there is little research on the effects of muscle fiber development and meat quality. Therefore, it is necessary to explore the mechanism of action of different fatty acid lengths on muscle fiber development. Therefore，108 healthy Megalobrama amblycephala with an initial average weight of (77.89±0.81) g were selected and randomly divided into 4 groups with 3 replicates each, and were fed with basal diet (control group) and butyric acid (BA), octanoic acid (OA) and palmitic acid (PA) instead of 1.5% soybean oil for 8 weeks. The results showed that the addition of fatty acids with different carbon-chain lengths had no significant effect on growth, nevertheless, it could significantly improve the adhesiveness of muscle, the number of muscle fibers, the proportion of smaller muscle fibers (<20 μm) and sarcomere length compared to the control group. In terms of gene expression, OA significantly increased the mRNA expression of the myogenic factor myf5. BA can up-regulate the mRNA expression of ampkα2 and sirt1 significantly, while down-regulating the myo-suppressor gene mstnb. Camk, the key gene of Ca²⁺-dependent signaling pathway, was also significantly elevated with the increase of the carbon-chain lengths of fatty acids in feed. This study showed that the dietary fatty acids with different carbon-chain lengths can effectively achieve the enhancement of muscle fibers development and meat quality of M. amblycephala through AMPK and Ca²⁺-dependent signaling pathways. This experiment provides basic data and theoretical basis for nutrient regulation of fish meat quality, and also a new pattern for nutritional enhancement of fish meat quality.