Bumblebee (Bombus spp.) venom contains a variety of components, including bombolitin, phospholipase A(2) (PLA(2)), serine proteases, and serine protease inhibitors. In this study, we identified a bumblebee (Bombus terrestris) venom serine protease inhibitor (Bt-KTI) that acts as a plasmin inhibitor. Bt-KTI consists of a 58-amino acid mature peptide that displays features consistent with snake venom Kunitz-type inhibitors, including six conserved cysteine residues and a P1 site. Recombinant Bt-KTI was expressed as a 6.5-kDa peptide in baculovirus-infected insect cells. The recombinant peptide demonstrated properties similar to Kunitz-type trypsin inhibitors. Bt-KTI showed no detectable inhibitory effects on factor Xa, thrombin, or tissue plasminogen activator; however, Bt-KTI strongly inhibited plasmin, indicating that it acts as an antifibrinolytic agent. These findings demonstrate the antifibrinolytic role of Bt-KTI as a plasmin inhibitor. Copyright © 2012 Elsevier Ltd. All rights reserved.