Abstract: A DNA fragment encoding rabbit defensin NP2 was cloned based on the published amino acid sequence and the biased codon usage of Pichia Pastoris. The modified rabbit defensin NP2 DNA fragment was inserted into Pichia Pastoris expression vector pGAPZα. Then the plasmid pGAPZαA- NP2 was transferred to E. coli JM109 and used for Pichia pastoris transformation. The recombinant linearized with restriction enzyme BlnI was transformed into special strain Pichia pastoris SMD1168. The positive clones were selected with PCR, and the recombinant rabbit defensin NP2 was detected with SDS-PGAE, and quantified with Bradford and then used for biological activity assay. The results show that the modified rabbit defensin NP2 can express in the Pichia pastoris, with the well ability to inhibit the growth of E. coli 5α. These findings have provided valuable tools for further studies on the function and medicinal application of NP2.