Antilipopolysaccharide factors (ALFs) are small basic proteins that can bind and neutralize lipopolysaccharide (LPS) and have broad spectrum antimicrobial activities. In this study, we describe the isolation of the full-length cDNA encoding for ALF peptide (ALFSp) of mud crab, Scylla paramamosain by sequencing a hemocyte cDNA library and using the rapid amplification cDNA end (RACE) method. A full-length ALFSp cDNA of 614 bp contains an open reading frame (ORF) of 372 bp, encoding 123 amino acid protein with 26 residues signal sequence. The calculated molecular mass of the mature protein is 11.18 kDa. The highly two conserve cysteine residues and putative LPS binding domain were observed in ALFSp peptide. Comparison of amino acid sequences revealed that ALFSp shared high identity with other known ALFs and had an overall similarity of 65, 64, 63, 61 and 59% to those of Fenneropenaeus chinensis, Litopenaeus vannamei, Marsupenaeus japonicus, Limulus polyphemus, and Tachypleus tridentatus, respectively. A neighbour-joining tree showed a clear differentiation of each species and also indicated that ALF from S. paramamosain, Carcinus maenas and Callinectes sapidus are closely related phylogenetically. The genomic DNA sequence of ALFSp gene consists of 1075 bp containing three exons and two introns. Tissue distribution analysis revealed that ALFSp was abundantly expressed in hemocytes, intestine, and muscle but not in eyestalk. The synthetic ALFSp peptide containing putative LPS binding domain revealed a strong antimicrobial activity against several bacteria especially on the growth of Gram-positive bacteria, Micrococcus luteus and Gram-negative bacteria, Vibrio harveyi suggested that ALFSp could play an essential role in defense mechanism in S. paramamosain.