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Staphylococcus aureusis an important pathogen implicated in various diseases, including staphylococcal food poisoning. Bacteriocins are considered safe and effective antimicrobial substances for the prevention of the growth of pathogenic bacteria. In this article, we describe the purification and characterization of pasteuricin, a novel bacteriocin produced by Staphylococcus pasteuriRSP-1. A cell-free supernatant of S. pasteuriRSP-1 exerted strong antimicrobial activity against staphylococci, including methicillin-resistant S. aureus(MRSA), and gram-positive bacteria. The loss of antimicrobial activity upon treatment with proteolytic enzymes confirmed the proteinaceous nature of pasteuricin. A rapid and pronounced bactericidal effect of pasteuricin was confirmed by a live-dead bacterial viability assay. To our knowledge, pasteuricin is the first reported S. pasteuribacteriocin that inhibits S. aureus. Because pasteuricin is characterized by strong antimicrobial activity and high stability, it has potential as an alternative antimicrobial agent to antibiotics.