Several diastereomers and an enantiomer of KKVVFKVKFKK, an antimicrobial peptide that acts on the lipid membrane of pathogens were synthesized to investigate the effect of D-amino acid substitution on stability, secondary structure, and activity. The stability of the peptide in serum was improved greatly by the D-amino acid substitutions. D-Amino acid substitutions at the N- and/or C-terminal of the peptide, which had little effect on the alpha-helical structure, and all D-amino acid substitutions that formed a left-handed alpha-helix maintained antimicrobial activity, whereas D-amino acid substitutions in the middle of the amino acid sequence disrupted the alpha-helical structure, resulting in the complete loss of activity. This result confirmed that the peptide did not interact with chiral receptors, enzymes, or any chiral component of the membrane. D-Amino acid substitutions at the termini reduced the inhibition of the activity by heat-inactivated serum, which indicated that local change of chirality or change of secondary structure induced by D-amino acid substitutions might affect the interactions between the peptide and certain components in the serum. The present study suggests that partial D-amino acid substitution is a useful technique to improve the in vivo activity of antimicrobial peptides.