Antimicrobial peptides (AMPs) comprise molecules that involve in the defense mechanism of various organisms towards pathogens such as bacteria, fungi, parasites and viruses. Crustins are generally defined as multi-domain cationic antimicrobial peptides containing one whey acidic protein (WAP) domain at the C-terminus as the functional unit. In this study, we identified and characterized a novel crustin homolog (Fi-Crustin2) with 354 bp fragment cDNA encoding 117 amino acids and an ORF of 100 amino acids with a net charge of +1 from the mRNA of F. indicus haemocytes. This study forms the second report of a crustin isoform from F. indicus. Blast analysis revealed that Fi-crustin2 exhibits similarity to shrimp crustins already reported. The active mature peptide has a molecular weight of 10.61 kDa and pI of 7.59 with a beta sheeted structure. The mature peptide was cloned into pET-32a(+) with a N-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli, and the recombinant crustin, Fi-crustin2 inhibited the growth of Gram-negative bacteria with low MIC. All these features suggest that Fi-crustin2 is a potent antibacterial protein against Gram-negative bacteria and could play an important role in the innate immune mechanism of F. indicus.