Myostatin (MSTN) gene was isolated from Japanese flounder(Paralichthys olivaceus) by homology cloning strategy and genome walking .By sequence analysis and verified in cDNA level, Japanese flounder MSTN gene included three exons and two introns, encoding a 374-amino-acid protein. Eight TATA box, one CAAT box and six E box located in 5` flanking region. A polyA signal existed in 3` flanking region. Nine conserved cysteine residues and an RXXR proteolytic cleavage domain were detected in the C terminal of protein. MSTN in Japanese flounder was high homologous with MSTN genes in fish via phylogeny analysis. The RT-PCR analysis demonstrated that MSTN was undetectable during embryo development and its transcription was influenced by environment factors in adult.