The study isolated and characterized a novel human antimicrobial peptide, human β-defensin-3 (hBD-3), from human lesional psoriatic scales and keratinocytes. hBD-3 is a 5-kDa, nonhemolytic peptide with broad-spectrum antimicrobial activity against various pathogenic microbes, including multiresistant *Staphylococcus aureus* and vancomycin-resistant *Enterococcus faecium*. Ultrastructural analysis revealed cell wall perforation in *S. aureus* treated with hBD-3. Recombinant hBD-3 expressed in *Escherichia coli* and chemically synthesized hBD-3 showed identical antimicrobial activity and biochemical properties to the natural peptide. hBD-3 was predominantly 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 crucial role in innate epithelial defense against infections by various microorganisms, particularly in skin and lung conditions like cystic fibrosis.The study isolated and characterized a novel human antimicrobial peptide, human β-defensin-3 (hBD-3), from human lesional psoriatic scales and keratinocytes. hBD-3 is a 5-kDa, nonhemolytic peptide with broad-spectrum antimicrobial activity against various pathogenic microbes, including multiresistant *Staphylococcus aureus* and vancomycin-resistant *Enterococcus faecium*. Ultrastructural analysis revealed cell wall perforation in *S. aureus* treated with hBD-3. Recombinant hBD-3 expressed in *Escherichia coli* and chemically synthesized hBD-3 showed identical antimicrobial activity and biochemical properties to the natural peptide. hBD-3 was predominantly 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 crucial role in innate epithelial defense against infections by various microorganisms, particularly in skin and lung conditions like cystic fibrosis.