<jats:title>Summary</jats:title><jats:p>Jadomycin production is under complex regulation in <jats:italic><jats:styled-content style="fixed-case">S</jats:styled-content>treptomyces venezuelae</jats:italic>. Here, another cluster‐situated regulator, <jats:styled-content style="fixed-case">JadR</jats:styled-content>*, was shown to negatively regulate jadomycin biosynthesis by binding to four upstream regions of <jats:styled-content style="fixed-case"><jats:italic>jadY</jats:italic></jats:styled-content>, <jats:styled-content style="fixed-case"><jats:italic>jadR1</jats:italic></jats:styled-content>, <jats:styled-content style="fixed-case"><jats:italic>jadI</jats:italic></jats:styled-content> and <jats:styled-content style="fixed-case"><jats:italic>jadE</jats:italic></jats:styled-content> in <jats:styled-content style="fixed-case"><jats:italic>jad</jats:italic></jats:styled-content> gene cluster respectively. The transcriptional levels of four target genes of <jats:styled-content style="fixed-case">JadR</jats:styled-content>* increased significantly in Δ<jats:styled-content style="fixed-case">jadR</jats:styled-content>*, confirming that these genes were directly repressed by <jats:styled-content style="fixed-case">JadR</jats:styled-content>*. Jadomycin <jats:styled-content style="fixed-case">B</jats:styled-content> (<jats:styled-content style="fixed-case">JdB</jats:styled-content>) and its biosynthetic intermediates 2,3‐dehydro‐<jats:styled-content style="fixed-case">UWM</jats:styled-content>6 (<jats:styled-content style="fixed-case">DHU</jats:styled-content>), dehydrorabelomycin (<jats:styled-content style="fixed-case">DHR</jats:styled-content>) and jadomycin <jats:styled-content style="fixed-case">A</jats:styled-content> (<jats:styled-content style="fixed-case">JdA</jats:styled-content>) modulated the <jats:styled-content style="fixed-case">DNA</jats:styled-content>‐binding activities of <jats:styled-content style="fixed-case">JadR</jats:styled-content>* on the <jats:styled-content style="fixed-case"><jats:italic>jadY</jats:italic></jats:styled-content> promoter, with <jats:styled-content style="fixed-case">DHR</jats:styled-content> giving the strongest dissociation effects. Direct interactions between <jats:styled-content style="fixed-case">JadR</jats:styled-content>* and these ligands were further demonstrated by surface plasmon resonance, which showed that <jats:styled-content style="fixed-case">DHR</jats:styled-content> has the highest affinity for <jats:styled-content style="fixed-case">JadR</jats:styled-content>*. However, only <jats:styled-content style="fixed-case">DHU</jats:styled-content> and <jats:styled-content style="fixed-case">DHR</jats:styled-content> could induce the expression of <jats:styled-content style="fixed-case"><jats:italic>jadY</jats:italic></jats:styled-content> and <jats:styled-content style="fixed-case"><jats:italic>jadR</jats:italic></jats:styled-content><jats:italic>* in vivo</jats:italic>. <jats:styled-content style="fixed-case">JadY</jats:styled-content> is the <jats:styled-content style="fixed-case">FMN</jats:styled-content>/<jats:styled-content style="fixed-case">FAD</jats:styled-content> reductase supplying cofactors <jats:styled-content style="fixed-case">FMNH</jats:styled-content><jats:sub>2</jats:sub>/<jats:styled-content style="fixed-case">FADH</jats:styled-content><jats:sub>2</jats:sub> for <jats:styled-content style="fixed-case">JadG</jats:styled-content>, an oxygenase, that catalyses the conversion of <jats:styled-content style="fixed-case">DHR</jats:styled-content> to <jats:styled-content style="fixed-case">JdA</jats:styled-content>. Therefore, our results revealed that <jats:styled-content style="fixed-case">JadR</jats:styled-content>* and early pathway intermediates, particularly <jats:styled-content style="fixed-case">DHR</jats:styled-content>, regulate cofactor supply by a convincing case of a feed‐forward mechanism. Such delicate regulation of expression of <jats:styled-content style="fixed-case"><jats:italic>jadY</jats:italic></jats:styled-content> could ensure a timely supply of cofactors <jats:styled-content style="fixed-case">FMNH</jats:styled-content><jats:sub>2</jats:sub>/<jats:styled-content style="fixed-case">FADH</jats:styled-content><jats:sub>2</jats:sub> for jadomycin biosynthesis, and avoid unnecessary consumption of <jats:styled-content style="fixed-case">NAD</jats:styled-content>(<jats:styled-content style="fixed-case">P</jats:styled-content>)<jats:styled-content style="fixed-case">H</jats:styled-content>.