A novel antimicrobial peptide, eumenitin, was isolated from the venom of the solitary eumenine wasp Eumenes rubronotatus. The sequence of eumenitin, Leu-Asn-Leu-Lys-Gly-Ile-Phe-Lys-Lys-Val-Ala-Ser-Leu-Leu-Thr, was mostly analyzed by mass spectrometry together with Edman degradation, and corroborated by solid-phase synthesis. This peptide has characteristic features of cationic linear alpha-helical antimicrobial peptides, and therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the CD spectra of eumenitin in the presence of TFE or SDS showed a high content of alpha-helical conformation. Eumenitin exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria, and moderately stimulated degranulation from the rat peritoneal mast cells and the RBL-2H3 cells, but showed no hemolytic activity against human erythrocytes. This antimicrobial peptide in the eumenine wasp venom may play a role in preventing potential infection by microorganisms during prey consumption by their larvae.