Hispidalin is a novel antimicrobial peptide isolated from the seeds of Benincasa hispida and is reported to have broad antimicrobial activity against various bacterial and fungal pathogens. To produce significant amounts of Hispidalin, a recombinant Hispidalin with an N-terminal 6 x His tag and an enterokinase sequence, for the first time, was successfully expressed in Escherichia coli or Pichia pastoris cell factory. Results showed that the E. coliderived recombinant Hispidalin did not show any antimicrobial activity against all the tested strains, whereas the P. pastoris-derived recombinant Hispidalin (rHispidalin) showed a broad antibacterial spectrum against five pathogenic bacteria of both Gram-negative and Gram-positive. rHispidalin also has bactericidal activity and completely killed all of the Staphylococcus aureus within 40 min. Additionally, rHispidalin showed a broad range of thermostability and pH stability, and a hemolytic activity of less than 2% even at a concentration of 300 mu g/ml; it was resistant to trypsin and proteinase K, but was moderately sensitive to pepsin and papain. Moreover, rHispidalin effectively permeabilized the cytoplasmic membrane and disrupted the morphology of targeted bacterial cells. After an initial optimization was performed, the amount of rHispidalin accumulation could reach as high as 98.6 mu g/ml. These results indicate that Hispidalin could be produced on a large scale by P. pastoris and has a great potential to be utilized as a new antibacterial agent for further development.