Anti-lipopolysaccharide factors (ALF) are a group of small basic proteins which are released into the hemolymph as a result of rapid degranulation of hemocytes in response to bacterial lipopolysaccharide (LPS). In the present study, using a combined approach of degenerate and RACE PCR, the gene coding for Scylla serrata anti-lipopolysaccharide factor (SsALF) was cloned and characterized. The full-length SsALF cDNA sequence consists of 607 bp and encodes a polypeptide of 97 amino acids, constituting a molecular mass of 11172 Da with an estimated pI of 10.01. The SsALF protein showed upto 92% similarity with ALF from Scylla paramamosain and about 33-53% amino acid sequence identity with other known ALF sequences. SsALF protein sequence demonstrated the presence of two highly conserved cysteine residues and putative LPS binding domain. An in vivo expression study showed that SsALF mRNA was expressed predominantly in hemocytes, heart and muscle of healthy mud crabs. The recombinant form of SsALF protein (rSsALF) was expressed with a Histag, in Escherichia coli, using the pTriEx-4 Ek/LIC vector. The purified rSsALF protein demonstrated antimicrobial activity against both Gram-positive and Gram-negative bacteria. The recombinant protein was able to significantly neutralize LPS-induced expression on SsALF in vivo as demonstrated by real-time PCR. rSsALF was able to permeabilize artificial phospholipid membranes as demonstrated by calcein enclosed liposome model. These studies strongly suggest that SsALF is one among the important antimicrobial factors produced in the crab during a microbial invasion.