Closthioamide (CTA) is a unique symmetric nonribosomal peptide with six thioamide moieties that is produced by the Gram-positive obligate anaerobe Ruminiclostridium cellulolyticum. CTA displays potent inhibitory activity against important clinical pathogens, making it a promising drug candidate. Yet, the biosynthesis of this DNA gyrase-targeting antibiotic has remained enigmatic. Using a combination of genome mining, genome editing (targeted group II intron, CRISPR/Cas9), and heterologous expression, we show that CTA biosynthesis involves specialized enzymes for starter unit biosynthesis, amide bond formation, thionation, and dimerization. Surprisingly, CTA biosynthesis involves a novel thiotemplated peptide assembly line that markedly differs from known nonribosomal peptide synthetases. These findings provide the first insights into the biosynthesis of thioamide-containing nonribosomal peptides and offer a starting point for the discovery of related natural products.