Abstract: Malate dehydrogenase gene (mdh) is closely associated with muscle growth, regulating muscle fiber growth and type transformation by influencing skeletal muscle energy metabolism. This study aimed to investigate the function and role of mdh in Megalobrama amblycephala by using CRISPR/Cas9 to knock-out the mdh and examining the phenotype and gene expression in the resulting mutants. The findings demonstrated that CRISPR/Cas9 can effectively knock-out mdh in M. amblycephala, with mutants exhibiting significant phenotypic differences from the control group. Mutants were notably smaller, with reduced body weight, length, height and width compared to controls. qPCR results showed that the expression level of mdh in the mutant individuals was significantly lower than that in the control group. In addition, the expression of MyoG, MyHCⅡa decreased significantly in mutants with reduced mdh, while MyHCⅡb showed no significant change. All the results suggest that CRISPR/Cas9 can be used for efficient and rapid gene editing. It also proved that the mdh gene may plays a promoting role in the growth of muscle in M. amblycephala, and the knock-out of mdh will inhibit the growth. The results of this study provide a certain theoretical value for the study of growth traits, which is conducive to the rational application of mdh in molecular breeding of M. amblycephala.