Hepcidin, a novel gene encoded, 25 residue, 2-3 KDa cysteine rich cationic peptide synthesized in liver cells play an important role in iron metabolism in addition to its antimicrobial activity. In this study hepcidin cDNA expressed on hepatocytes of Bubalus bubalis has been characterized and the antibacterial activity of buffalo hepcidin analog has been determined. The complete buffalo hepcidin sequence is of 324 bp and have an ORF of 249 nucleotides from 14 to 262. The sequence analysis at nucleotide level showed homology of 98.0% with cattle, 84.3% with pig, whereas with human, chimpanzee and gorilla 80.3%, 80.7% and 81.5% respectively. The percent identity of buffalo hepcidin cDNA to that of japanese macaque, dog, gibbon, mouse and norway rat are81.9%, 79.9%, 79.9%, 67.9% and 65.9% respectively. The hepcidin cDNA of buffalo and cattle showed a total difference of five nucleotides at position 15, 108, 225, 240 and 246 with no difference in deduced amino acid sequence. The base A(15), C(108), A(225), C(240), A(246) in cattle are replaced by G(15), G(108), G(225), T(240) and G(246) respectively in buffalo. The deduced amino acid sequence of buffalo preprohepcidin consists of 82 amino acids containing 10 strongly basic, 3 strongly acidic, 27 hydrophobic and 29 polar amino acids. The predicted molecular weight and isoelectric point (P(I)) for the peptide is 8883.67 Daltons and 8.804 respectively. Phylogenetic analysis both at nucleotide and amino acid level indicate that buffalo and cattle hepcidin sequences comprises one clad which is more closely related to pig compared to human. The chemically synthesized analog of buffalo hepcidin using Fmoc chemistry in solid phase had antibacterial activity.