Papiliocin is a well-studied antimicrobial peptide that is isolated from the swallowtail butterfly, Papilio xuthus. Papiliocin is a novel antibiotic possessing low toxicity against mammalian cells and remarkable bacterial cell selectivity. To understand structure-activity relationships, the three dimensional structure of papiliocin was determined in 300 mM dodecylphosphocholine (DPC) micelles by NMR spectroscopy, showing that papiliocin has N-terminal α-helical structure from Lys3 to Lys21 linked to a hydrophobic C-terminal helix from Ala25 to Val36 by a hinge region. Interactions between papiliocin and LPS studied using tryptophan blue-shift data and STD-NMR experiments revealed that Trp2 and Phe5 at N-terminal helix play an essential role in attracting papiliocin into the cell membrane of Gram-negative bacteria. To investigate functional importance of N-terminal and C-terminal helix as well as the aromatic residues, five papiliocin analogs were designed. Two 22-mer analogs composed of N-terminal helix, PapN including N-terminal helix from Arg1 to Ala22 of papiliocin and PapN-FW with exchange of Trp2 and Phe5 in PapN, showed anti-inflammatory activity as well as antimicrobial activity against Gram-negative bacteria without toxicity against mammalian cells. Fluorescence dye leakage experiments revealed that Trp2 in PapN and Trp5 in PapN-FW play key roles in targeting the bacterial cytoplasmic membrane similar to Trp2 in papiliocin. Furthermore, hydrophobic interactions between the C-terminal helix of papiliocin and the hydrophobic portion of LPS or other Gram-negative bacteria cell components provide papiliocin with selectivity against Gram-negative bacterial membranes including LPS.