The influence of number of signal peptide cleavage site on extracellular production of recombinant cutinase was described in the present work. Firstly, recombinant Escherichia con BL/pET-CUT1 and BL/pET-CUT2 that expressed precursor cutinase with one and two signal peptide cleavage sites were constructed, respectively. Under the different culture condition, cutinase activity in periplasm and medium of BL/pET-CUT2 were higher than that of BL/pET-CUT1. When cultured in 30 C with glycerol of 10g/l, 85.2 U/ml cutinase activity was detected in periplasm of BL/pET-CUT2 after 40h cultivation, while 44.6 U/ml was found in BL/pET-CUT1. It also was found that the cutinase activity in culture medium of BL/pET-CUT 2 reached to 130.3 U/ml while the activity of BL/pIET-CUT 1 reached to 95.7 U/ml after 64h of culture. From this investigation we have found that the increase of signal peptide cleavage site can improve the extracellular production of recombinant cutinase in E. coli. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.