Gliotoxin is an important epipolythiodioxopiperazine, which was biosynthesized by the gli gene cluster in Aspergillus genus. However, the regulatory mechanism of gliotoxin biosynthesis remains unclear. In this study, a novel Zn2Cys6 transcription factor DcGliZ that is responsible for the regulation of gliotoxin biosynthesis from the deep-sea-derived fungus Dichotomomyces cejpii was identified. DcGliZ was expressed in Escherichia coli and effectively purified from inclusion bodies by refolding. Using electrophoretic mobility shift assay, we demonstrated that purified DcGliZ can bind to gliG, gliM, and gliN promoter regions in the gli cluster. Furthermore, the binding kinetics and affinity of DcGliZ protein with different promoters were measured by surface plasmon resonance assays, and the results demonstrated the significant interaction of DcGliZ with the gliG, gliM, and gliN promoters. These new findings would lay the foundation for the elucidation of future gliotoxin biosynthetic regulation mechanisms in D. cejpii.The putative gene cluster (gli) of gliotoxin is composed of 13 genes that encode crucial enzymes responsible for the biosynthesis of various gliotoxins and their derivatives in A. fumigatus [9]. The non-ribosomal peptide synthetase encoded by gliP gene catalyzes diketopiperazine scaffold formation, the first biosynthetic reaction in a gliotoxin biosynthesis pathway [10]. Subsequently, cytochrome P450 monooxygenase encoded by gliC catalyzes the hydroxylation at the α-carbon of L-Phe [11]. Then, glutathione S-transferase (GST) encoded by the gliG promotes the sulfurization of gliotoxin biosynthetic imtermediates; GST is known for its ability in the catalyzation of carbon-sulfur bond formation, as opposed to detoxification [12,13]. After the process by the enzymes of GliK and GliJ, the γ-glutamyl moieties are removed [14,15]. The carbon-sulfur lyase expressed by the gliI gene then catalyzes the intermediate to generate a notorious epidithiol moiety [16]. After the catalysis of cytochrome P450 monooxygenases (GliF or GliC) and GliH (function remains elusive), the N-methyltransferase encoded by gliN or gliM functions as a freestanding amide to promote amide methylation and confer stability on ETP [17,18]. Finally, the oxidoreductase GliT-mediated disulfide bridge closure might be a prerequisite for the formation of gliotoxins, and the major facilitator superfamily transporter GliA also plays an important role in exporting the toxins to prevent the harmful effect of gliotoxin on hosts [9,19].Many regulators involved in gliotoxin biosynthesis pathway have been identified in A. fumigatus, regardless of whether they deploy a non-gli cluster or a gli cluster. GtmA (or termed TtmA) is encoded outside the gli cluster and functions as a bis-thiomethyl transferase for the conversion of dithiogliotoxin to bisdethiobis (methylthio) gliotoxin (BmGT), which mainly attenuates the formation of disulfide bridge closure [20]. Other non-gli clusters encoding transcription factors, including the global regulator laeA, C 2 H 2...