<jats:title>ABSTRACT</jats:title> <jats:p> This research was initiated to search for novel antimicrobial compounds produced by food or environmental microorganisms. A new bacterial strain, designated OSY-SE, which produces a unique and potent antimicrobial agent was isolated from soil. The isolate was identified as a <jats:named-content content-type="genus-species">Paenibacillus</jats:named-content> sp. through cultural, biochemical, and genetic analyses. An antimicrobial compound was extracted from <jats:named-content content-type="genus-species">Paenibacillus</jats:named-content> OSY-SE with acetonitrile and purified using liquid chromatography. After analyses by mass spectrometry (MS) and nuclear magnetic resonance (NMR), the antimicrobial compound was determined to be a cyclic lipopeptide consisting of a C <jats:sub>15</jats:sub> fatty acyl (FA) chain and 13 amino acids. The deduced sequence is FA-Orn-Val-Thr-Orn-Ser-Val-Lys-Ser-Ile-Pro-Val-Lys-Ile. The carboxyl-terminal Ile is connected to Thr by ester linkage. The new compound, designated paenibacterin, showed antagonistic activities against most Gram-positive and Gram-negative bacteria tested, including <jats:named-content content-type="genus-species">Listeria monocytogenes</jats:named-content> , methicillin-resistant <jats:named-content content-type="genus-species">Staphylococcus aureus</jats:named-content> , <jats:named-content content-type="genus-species">Escherichia coli</jats:named-content> O157:H7, and <jats:named-content content-type="genus-species">Salmonella enterica</jats:named-content> serovar Typhimurium. Paenibacterin is resistant to trypsin, lipase, α-glucosidase, and lysozyme. Its antimicrobial activity was lost after digestion by pronase and polymyxin acylase. Paenibacterin is readily soluble in water and fairly stable to exposure to heat and a wide range of pH values. The new isolate and its antimicrobial agent are being investigated for usefulness in food and medical applications.