Verruculogen is a tremorgenic mycotoxin and contains an endoperoxide bond. In this study, we describe the cloning, overexpression and purification of a non-heme Fe(II) and α-ketoglutarate-dependent dioxygenase FtmOx1 from Aspergillus fumigatus, which catalyses the converstion of fumitremorgin B to verruculogen by inserting an endoperoxide bond between two prenyl moieties. Incubation with ¹⁸O₂-enriched atmosphere demonstrated that both oxygen atoms of the endoperoxide bond are derived from one molecule of O₂. FtmOx1 is the first endoperoxide-forming non-heme Fe(II) and α-ketoglutarate-dependent dioxygenase reported so far. A mechanism of FtmOx1-catalysed verruculogen formation is postulated and discussed.