We purified an antibacterial peptide from the larval hemolymph of a coleopteran beetle, Acalolepta luxuriosa. Using structure analysis and cDNA cloning, this peptide was identified as a novel member of the insect defensin family and named A. luxuriosa defensin 1. A. luxuriosa defensin 1 shares high sequence similarity with other defensins, especially coleopteran defensins. A dendrogram of coleopteran insect defensins based on sequence homology revealed that A. luxuriosa defensin is closer to Tenebrionoidea defensin than to Scarabaeoidea defensin, which parallels the evolutionary relationship of these coleopteran insects. Although A. luxuriosa defensin 1 was most homologous with Tenebrio molitor tenecin 1, its antibacterial spectrum was broader, affecting the growth of Gram-positive and Gram-negative bacteria, suggesting that the variability of the antibacterial spectrum results from small sequence differences.