Synonymous mutation can induce phenotypic variation by altering gene transcription, translation and post-transcription process in recent studies on human disease. To study the effects of synonymous mutation on gene transcription and translation, the total of 12 genotypes (CG, TA, 1T, 2T, 3T, 4A, 5T, 1C, 2C, 3C, 4G and 5G) deduced from the five SNP synonymously mutated loci in MSTN genes of Chinese mitten crab (Eriocheir sinensis) (Es-MSTN), such as SNP1(C/T), SNP2(C/T), SNP3(C/T), SNP4(A/G) and SNP5(T/G), were employed by the means of site-directed mutagenesis, in vitro transcription, and gene overexpression. The results showed that the 12 alleles displayed obviously different transcriptional efficiency in vitro, and the highest transcriptional level was found in the 1C allele and the lowest level in the CG and TA alleles, in which the TA allele exhibited significantly higher transcriptional level than the CG allele. The overexpression in 293T cells found that expression intensity of different mutant genotypes was significantly different at different time (24, 36, 48, 60, 72, 84, 96 and 108 h) after transfection, and the TA allele exhibited significantly higher expression level than the CG allele. Generally, the present study indicated that these five synonymous mutations had important effects on transcription and translation of Es-MSTN gene, suggesting that synonymous mutations could play an important role in biological process. Further studies at post-transcriptional and post-translational levels may shed light on the intrinsic relationship between gene function and phenotypic variation in E. sinensis.