This work reports, for the first time, the purification, characterization and antibacterial activity of an elastase inhibitor from Lasiodora sp. hemocytes (ElLaH). The hemocyte extract inhibited chymotrypsin (22%). trypsin (44%), tissue plasminogen activator (52%), urokinase (58%) and human neutrophil elastase (99%). ElLaH was purified by Trypsin-Sepharose column and RP-HPLC. SDS-PAGE of ElLaH revealed a molecular mass of 8 kDa and MALDI-TOF mass spectrometry revealed a single molecular mass of 8274 Da. The amino terminal sequence determined was LPC(PF)PYQQELTC. The dissociation constant (K) for human neutrophil elastase was 0.32 nM. Hemocyte extract exerted antibacterial effect on Bacillus subtilis and Enterococcus faecalis, while ElLaH was only active against E. faecalis. Currently, Lasiodora sp. is undergoing a systematic review and this study contributes to molecular characterization of the genus. In addition, the results suggest that serine protease inhibitors expressed in Lasiodora sp. hemocytes may be involved in the defense against bacterial infection. (C) 2011 Elsevier Ltd. All rights reserved.