high density fermentation technology for the production of antimicrobial peptide PSI in a 100 L fermentor using a recombinantKluyveromyces lactisstrain was reported in this paper. The effects of mediums; cell density; as well as the inducer lactose and its fed-batch models; on the production of PSI were investigated. Results showed that 5 kg inducer lactose should be added with feeding velocity of 1 L/h when the cell density reach 190 g/L during the fermentation using BSM medium. After 25 h of induction culture; the cell wet weight; the protein yield; and the titer of PSI reached 280 g/L; 27.8 mg/L; and 2.36×109U/L; respectively. The separation and purification of the PSI in the fermentation broth were also investigated. The purification efficiency of anion exchange chromatography; ultrafiltration; precipitation of ammonium sulphate or acetone were 8.83×109; 6.35×109; 4.56×109; and 3.28×109U/L; respectively; which indicated that the most efficient technique for purification of PSI was anion exchange chromatography.