Porcine β-defensin 2 (pBD2), a recently discovered porcine defensin that is produced by the intestine, exerts antimicrobial activities and innate immune effects that are linked to intestinal diseases in pigs.Here, we report a codon-optimised protein corresponding to mature pBD2 cDNA that was expressed and purified in Pichia pastoris yeast.The highest amount of secreted protein (3,694.0mg/L) was reached 144h into a 150-h induction during high density cultivation.Precipitation followed by gel exclusion chromatography yielded 383.7mg/L purified recombinant pBD2 (rpBD2) with a purity of 93.7%.Two recombinant proteins of 5,458.5 and 5,258.4Da were detected in the mass spectrum due to variation in the amino-terminus.The rpBD2 exhibited high antimicrobial activity against a broad range of pig pathogenic bacteria (minimal inhibitory concentration [MIC]: 32 ~ 128μg/mL); the highest activity was observed against Salmonella choleraesuis, Staphylococcus aureus and Streptococcus suis (MIC: 32 ~ 64μg/mL).However, rpBD2 also inhibited the growth of probiotics such as Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae, but at lower efficacies than the pathogens.Purified or unpurified rpBD2 also maintained high activity over a wide range ofpH values (2.0 ~ 10.0), a high thermal stability at 100℃ for 40 min, and significant resistance to papain, pepsin and trypsin.In addition, the activity of rpBD2 toward S.aureus was unaffected by 10 mM dithiothreitol (DTT) and 20% dimethyl sulphoxide (DMSO).Our results suggest that pBD2 could be produced efficiently in large quantities in P.pastoris, and be a substitute for traditional antibiotics for growth promotion in the porcine industry.