KR-12 (residues 18–29 of LL-37) was known to be the smallest peptide of human cathelicidin LL-37 possessing antimicrobial activity. In order to optimize α-helical short antimicrobial peptides having both antimicrobial and antiendotoxic activities without mammalian cell toxicity, we designed and synthesized a series of KR-12 analogs. Highest hydrophobic analogs KR-12-a5 and KR-12-a6 displayed greater inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α production and higher LPS-binding activity. We have observed that antimicrobial activity is independent of charge, but LPS neutralization requires a balance of hydrophobicity and net positive charge. Among KR-12 analogs, KR-12-a2, KR-12-a3 and KR-12-a4 showed much higher cell specificity for bacteria over erythrocytes and retained antiendotoxic activity, relative to parental LL-37. KR-12-a5 displayed the strongest antiendotoxic activity but almost similar cell specificity as compared with LL-37. Also, these KR-12 analogs (KR-12-a2, KR-12-a3, KR-12-a4 and KR-12-a5) exhibited potent antimicrobial activity (minimal inhibitory concentration: 4 μM) against methicillin-resistant Staphylococcus aureus. Taken together, these KR-12 analogs have the potential for future development as a novel class of antimicrobial and anti-inflammatory therapeutic agents. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.