A novel human inducible antimicrobial peptide, human β-defensin-3 (hBD-3), was isolated from human psoriatic scales and cloned from keratinocytes. hBD-3 demonstrated broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, including multiresistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. It showed no hemolytic activity and was indistinguishable from naturally occurring peptide in antimicrobial activity and biochemical properties. hBD-3 was expressed in skin and tonsils, with keratinocytes and airway epithelial cells as major sources. Tumor necrosis factor α and bacterial contact induced hBD-3 mRNA expression. hBD-3 may play a role in innate epithelial defense against infections in skin and lung, such as cystic fibrosis. The study also showed that hBD-3 could be expressed as a His-Tag fusion protein in E. coli and chemically synthesized. Recombinant and synthetic hBD-3 exhibited identical antimicrobial activity and biochemical properties to the naturally occurring peptide. Ultrastructural analysis of hBD-3-treated S. aureus revealed signs of cell wall perforation. The study confirmed that hBD-3 is a broad-spectrum antimicrobial peptide, not salt-sensitive, and is a novel member of the β-defensin family. The findings suggest that hBD-3 could be important in the innate immune response to infections in skin and lung.A novel human inducible antimicrobial peptide, human β-defensin-3 (hBD-3), was isolated from human psoriatic scales and cloned from keratinocytes. hBD-3 demonstrated broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, including multiresistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. It showed no hemolytic activity and was indistinguishable from naturally occurring peptide in antimicrobial activity and biochemical properties. hBD-3 was expressed in skin and tonsils, with keratinocytes and airway epithelial cells as major sources. Tumor necrosis factor α and bacterial contact induced hBD-3 mRNA expression. hBD-3 may play a role in innate epithelial defense against infections in skin and lung, such as cystic fibrosis. The study also showed that hBD-3 could be expressed as a His-Tag fusion protein in E. coli and chemically synthesized. Recombinant and synthetic hBD-3 exhibited identical antimicrobial activity and biochemical properties to the naturally occurring peptide. Ultrastructural analysis of hBD-3-treated S. aureus revealed signs of cell wall perforation. The study confirmed that hBD-3 is a broad-spectrum antimicrobial peptide, not salt-sensitive, and is a novel member of the β-defensin family. The findings suggest that hBD-3 could be important in the innate immune response to infections in skin and lung.