Abstract: Smad4 is a key protein in TGF-β signal transduction, which can regulate the developmental processes like cell proliferation and differentiation. In order to study the role of Smad4 in developmental process of grass carp(Ctenopharyngodon idellus), a full length of 2 974 bp of Smad4 cDNA sequence from grass carp was obtained, a 1 644 bp CDS (coding sequence) encoding a polypeptide of 547 amino acids was determined in the Smad4 of grass carp. Bioinformatics analysis showed that the relative molecular mass of grass carp Smad4 protein was 59 690.3, the molecular formula was C_{2 632}H_{4 073}N₇₅₃O₇₉₁S₂₄, isoelectric point was 6.5, and it contained both MH1 and MH2 highly conserved functional domains. BLAST homology analysis showed higher homology of grass carp Smad4 protein with common carp and zebrafish, which was 94.23% and 92.97% respectively. The open reading frame of grass carp Smad4 gene was reversely inserted into the expression vector pcDNA3.1(+), which constructed the antisense Smad4 gene eukaryotic expression vector pcDNA31(+)-Anti-Smad4.