Three major components of bumblebee venom are bombolitin, phospholipase A₂, and a serine protease, with bombolitin being the most abundant. Here, we describe the molecular cloning of bombolitin isolated from the venom of a bumblebee, Bombus terrestris, and demonstrate its antibacterial activity. The B. terrestris bombolitin gene consists of 2 exons encoding 56 amino acid residues. Comparative analysis shows that mature B. terrestris bombolitin consists of 18 amino acid residues, which are identical to those of B. ignitus bombolitin. B. terrestris bombolitin displayed antibacterial activity against both the Gram-negative bacterium Klebsiella pneumoniae and the Gram-positive bacterium Staphylococcus aureus, indicating that B. terrestris bombolitin may be a potential antimicrobial agent.