Defensins are small antimicrobial peptides that play an important role in the innate immune system of mammals. Here, we describe the antimicrobial activity of pBD-2, a recently discovered new porcine defensin that is produced in the intestine. A synthetic peptide corresponding to the mature protein showed high antimicrobial activity against a broad range of pathogenic bacteria, while it only showed limited hemolytic activity against porcine red blood cells. Highest activity was observed against Salmonella typhimurium, Listeria monocytogenes and Erysipelothrix rhusiopathiae. pBD-2 (4-8microM) killed these pathogens within 3h. The activity of pBD-2 against S. typhimurium was studied in more detail. At the minimum bactericidal concentration (MBC) of pBD-2, complete killing of S. typhimurium was relatively fast with no viable bacteria left after 90 min. However, antimicrobial activity of pBD-2 was decreased at higher ionic strengths with no residual activity at 150mM NaCl. Transmission electron microscopy of pBD-2 treated S. typhimurium indicated that relatively low doses of pBD-2 caused a retraction of the cytoplasmic membrane, while pBD-2 concentrations close to the MBC led to cytoplasm leakage and complete lysis of bacterial cells. Considering the site of production and the activity, pBD-2 may be an important defense molecule for intestinal health.