The recombinant defensin PaDef was for the first time expressed in Pichia pastoris. The optimal culture condition for recombinant PaDef expression was determined. The recombinant PaDef was produced with a yield of 79.6 μg/ml medium. The recombinant PaDef inhibited both gram-positive and gram-negative bacteria. Escherichia coli (ATCC 10305) was extremely sensitive to the recombinant PaDef.