The hydroxamate moiety of the natural product actinonin mediates inhibition of metalloproteinases because of its chelating properties towards divalent cations in the active site of those enzymes. Due to its antimicrobial activity actinonin has served as a lead compound for the development of new antibiotic drug candidates. Recently, we identified a putative gene cluster for the biosynthesis of actinonin. Here, we confirm and characterize this cluster by heterologous pathway expression and gene deletion experiments. We were able to assign the biosynthetic gene cluster to actinonin production and determine the cluster boundaries. Furthermore, we could establish ActI, an AurF‐like oxygenase, to be responsible for the N‐hydroxylation reaction that forms the hydroxamate warhead. Our findings provide the basis for more detailed investigations of actinonin biosynthesis.