<jats:title>ABSTRACT</jats:title> <jats:p> Operons are multigene transcriptional units which occur mostly in prokaryotes but rarely in eukaryotes. Protein-coding operons have not been reported in the Fungi even though they represent a very diverse kingdom of organisms. Here, we report a functional operon involved in the secondary metabolism of the fungus <jats:italic>Glarea lozoyensis</jats:italic> belonging to Leotiomycetes (Ascomycota). Two contiguous genes, <jats:italic>glpks3</jats:italic> and <jats:italic>glnrps7</jats:italic> , encoding polyketide synthase and nonribosomal peptide synthetase, respectively, are cotranscribed into one dicistronic mRNA under the control of the same promoter, and the mRNA is then translated into two individual proteins, GLPKS3 and GLNRPS7. Heterologous expression in <jats:named-content content-type="genus-species">Aspergillus nidulans</jats:named-content> shows that the GLPKS3-GLNRPS7 enzyme complex catalyzes the biosynthesis of a novel pyrrolidinedione-containing compound, xenolozoyenone (compound 1), which indicates the operon is functional. Although it is structurally similar to prokaryotic operons, the <jats:italic>glpks3-glnrps7</jats:italic> operon locus has a monophylogenic origin from fungi rather than having been horizontally transferred from prokaryotes. Moreover, two additional operons, <jats:italic>glpks28-glnrps8</jats:italic> and <jats:italic>glpks29-glnrps9</jats:italic> , were verified at the transcriptional level in the same fungus. This is the first report of protein-coding operons in a member of the Fungi. <jats:p> <jats:bold>IMPORTANCE</jats:bold> Operons are multigene transcriptional units which occur mostly in prokaryotes but rarely in eukaryotes. Three operon-like gene structures for secondary metabolism that were discovered in the filamentous fungus <jats:italic>Glarea lozoyensis</jats:italic> are the first examples of protein-coding operons identified in a member of the Fungi. Among them, the <jats:italic>glpks3-glnrps7</jats:italic> operon is responsible for the biosynthesis of xenolozoyenone, which is a novel tetramic acid-containing compound. Although structurally similar to prokaryotic operons, the <jats:italic>glpks3-glnrps7</jats:italic> operon locus did not result from horizontal gene transfer from prokaryotes. In addition, operonlike structures have been predicted <jats:italic>in silico</jats:italic> to be common in other fungi. The common occurrence and operonlike structure in fungi provide evolutionary insight and essential data for eukaryotic gene transcription.