<jats:p>Cyclothiazomycin is a member of the thiopeptide antibiotics, which are usually complicated derivatives of ribosomally synthesized peptides. A gene cluster containing 12 ORFs identical to the<jats:italic>clt</jats:italic>cluster encoding cyclothiazomycin from<jats:italic>Streptomyces hygroscopicus</jats:italic>10-22 was revealed by genome sequencing in<jats:italic>S. hygroscopicus</jats:italic>5008. Genes<jats:italic>SHJG8833</jats:italic>and<jats:italic>SHJG8837</jats:italic>of the cluster and flanking gene<jats:italic>SHJG8838</jats:italic>were predicted to encode regulatory proteins from different families. In this study, we showed that the newly identified cluster is functional and we investigated the roles of these regulatory genes in the regulation of cyclothiazomycin biosynthesis. We determined that<jats:italic>SHJG8833</jats:italic>, but not<jats:italic>SHJG8837</jats:italic>or<jats:italic>SHJG8838</jats:italic>, is critical for cyclothiazomycin biosynthesis. The transcriptional start point of<jats:italic>SHJG8833</jats:italic>was located to a thymidine 54 nt upstream of the start codon. Inactivation of<jats:italic>SHJG8833</jats:italic>abrogated the production of cyclothiazomycin, and synthesis could be restored by reintroducing<jats:italic>SHJG8833</jats:italic>into the mutant strain. Gene expression analyses indicated that SHJG8833 regulates a consecutive set of seven genes from<jats:italic>SHJG8826</jats:italic>to<jats:italic>SHJG8832</jats:italic>, whose products are predicted to be involved in different steps in the construction of the main framework of cyclothiazomycin. Transcriptional analysis indicated that these seven genes may form two operons,<jats:italic>SHJG8826</jats:italic>–<jats:italic>27</jats:italic>and<jats:italic>SHJG8828</jats:italic>–<jats:italic>32</jats:italic>. Gel-shift analysis demonstrated that the DNA-binding domain of SHJG8833 binds the promoters of<jats:italic>SHJG8826</jats:italic>and<jats:italic>SHJG8828</jats:italic>and sequences internal to<jats:italic>SHJG8826</jats:italic>and<jats:italic>SHJG8829</jats:italic>, and a conserved binding sequence was deduced. These results indicate that SHJG8833 is a positive regulator that controls cyclothiazomycin biosynthesis by activating structural genes in the<jats:italic>clt</jats:italic>cluster.