Perinerin, an antimicrobial peptide (GenBank No. P84117), was isolated and characterized from Asian marine clamworms, Perinereis aibuhitensis Grube. This peptide has effects against both gram-positive and gram-negative bacteria in vitro, especially on Pseudemonas aeruginosa. In our study, bioactive perinerin was expressed in Pichia pastoris, and characterized its physicochemical properties. The expressed sample was firstly analyzed by Tricine-SDS-PAGE, after then the recombinant proteins were purified by 2 kD MW cut-off (MWCO) ultrafiltration membrane, and finally purified by 10 kD MWCO ultrafiltration membrane and CM 52-ion exchange chromatography. About 6% protein was obtained by this so called three-purification method. Our results showed that Pichia pastoris was a suitable system for secreting perinerin. Bioactivity assay proved that the recombinant perinerin had antimicrobial effects.