<jats:title>ABSTRACT</jats:title> <jats:p> Polymyxin, a long-known peptide antibiotic, has recently been reintroduced in clinical practice because it is sometimes the only available antibiotic for the treatment of multidrug-resistant gram-negative pathogenic bacteria. Lack of information on the biosynthetic genes of polymyxin, however, has limited the study of structure-function relationships and the development of improved polymyxins. During whole genome sequencing of <jats:italic>Paenibacillus polymyxa</jats:italic> E681, a plant growth-promoting rhizobacterium, we identified a gene cluster encoding polymyxin synthetase. Here, we report the complete sequence of the gene cluster and its function in polymyxin biosynthesis. The gene cluster spanning the 40.6-kb region consists of five open reading frames, designated <jats:italic>pmxA</jats:italic> , <jats:italic>pmxB</jats:italic> , <jats:italic>pmxC</jats:italic> , <jats:italic>pmxD</jats:italic> , and <jats:italic>pmxE</jats:italic> . The <jats:italic>pmxC</jats:italic> and <jats:italic>pmxD</jats:italic> genes are similar to genes that encode transport proteins, while <jats:italic>pmxA</jats:italic> , <jats:italic>pmxB</jats:italic> , and <jats:italic>pmxE</jats:italic> encode polymyxin synthetases. The insertional disruption of <jats:italic>pmxE</jats:italic> led to a loss of the ability to produce polymyxin. Introduction of the <jats:italic>pmx</jats:italic> gene cluster into the <jats:italic>amyE</jats:italic> locus of the <jats:italic>Bacillus subtilis</jats:italic> chromosome resulted in the production of polymyxin in the presence of extracellularly added <jats:sc>l</jats:sc> -2,4-diaminobutyric acid. Taken together, our findings demonstrate that the <jats:italic>pmx</jats:italic> gene cluster is responsible for polymyxin biosynthesis.