<jats:title>ABSTRACT</jats:title> <jats:p> Thiopeptide antibiotics are an important class of natural products resulting from posttranslational modifications of ribosomally synthesized peptides. Cyclothiazomycin is a typical thiopeptide antibiotic that has a unique bridged macrocyclic structure derived from an 18-amino-acid structural peptide. Here we reported cloning, sequencing, and heterologous expression of the cyclothiazomycin biosynthetic gene cluster from <jats:italic>Streptomyces hygroscopicus</jats:italic> 10-22. Remarkably, successful heterologous expression of a 22.7-kb gene cluster in <jats:italic>Streptomyces lividans</jats:italic> 1326 suggested that there is a minimum set of 15 open reading frames that includes all of the functional genes required for cyclothiazomycin production. Six genes of these genes, <jats:italic>cltBCDEFG</jats:italic> flanking the structural gene <jats:italic>cltA</jats:italic> , were predicted to encode the enzymes required for the main framework of cyclothiazomycin, and two enzymes encoded by a putative operon, <jats:italic>cltMN</jats:italic> , were hypothesized to participate in the tailoring step to generate the tertiary thioether, leading to the final cyclization of the bridged macrocyclic structure. This rigorous bioinformatics analysis based on heterologous expression of cyclothiazomycin resulted in an ideal biosynthetic model for us to understand the biosynthesis of thiopeptides.