NK-lysin is an effector protein of the innate immune system, an important component of host protection. NK-lysin is a cationic anti-bacterial peptide, and is composed of five amphipathic α-helices. Their primary sequences are rich in positively charged amino acids and have six conserved cysteines that make three intra-disulfide bonds. We isolated a NK-lysin SNP between different chicken breeds. The A to G transition at position 271 nucleotide in the ORF results in an N to D amino acid change in the protein. We synthesized two 30 aa peptides to compare NK-lysin biological activity resulting from the SNP causing missense mutation (N91D). CD spectroscopy of the two synthetic peptides N91 and N91D in a liposomal solution showed spectra typical of α-helical peptides, but helical stability of N91D was reduced substantially when compared to that of N91. The N91 peptide exhibited greater anti-bacterial and anti-cancer activity than the N91D peptide. These results suggest that differences in electrostatic interaction between the helical peptide and lipid membrane results from the SNP and is a major factor in anti-bacterial and anti-cancer activity.