<jats:title>Summary</jats:title><jats:p>Two regulatory genes, <jats:styled-content style="fixed-case"><jats:italic>jadR2</jats:italic></jats:styled-content> and <jats:styled-content style="fixed-case"><jats:italic>jadR3</jats:italic></jats:styled-content>, in the jadomycin (<jats:styled-content style="fixed-case"><jats:italic>jad</jats:italic></jats:styled-content>) biosynthetic gene cluster of <jats:styled-content style="fixed-case"><jats:italic>S</jats:italic></jats:styled-content><jats:italic>treptomyces venezuelae</jats:italic> encode homologues of γ‐butyrolactone receptor. JadR2 was previously shown to be a pseudo γ‐butyrolactone receptor. <jats:styled-content style="fixed-case"><jats:italic>jadR3</jats:italic></jats:styled-content> is situated at the upstream of <jats:styled-content style="fixed-case"><jats:italic>jadW123</jats:italic></jats:styled-content> encoding putative enzymes for γ‐butyrolactone biosynthesis. Disruption of <jats:styled-content style="fixed-case"><jats:italic>jadR3</jats:italic></jats:styled-content> resulted in markedly decreased production of jadomycin. Transcriptional analysis revealed that JadR3 represses <jats:styled-content style="fixed-case"><jats:italic>jadW1</jats:italic></jats:styled-content>, <jats:styled-content style="fixed-case"><jats:italic>jadR2</jats:italic></jats:styled-content> and <jats:styled-content style="fixed-case"><jats:italic>jadR3</jats:italic></jats:styled-content> but activates <jats:styled-content style="fixed-case"><jats:italic>jadR1</jats:italic></jats:styled-content>, the key activator gene for jadomycin biosynthesis. <jats:styled-content style="fixed-case">DNase I</jats:styled-content> footprinting showed that JadR3 has four binding sites in the intergenic regions of <jats:styled-content style="fixed-case"><jats:italic>jadR2–jadR1</jats:italic></jats:styled-content> and <jats:styled-content style="fixed-case"><jats:italic>jadR3–jadW1</jats:italic></jats:styled-content>. A <jats:styled-content style="fixed-case">JadR3</jats:styled-content> interactive molecule, <jats:styled-content style="fixed-case">SVB1</jats:styled-content>, was purified from a large‐scale fermentation and its structure found to be the same as <jats:styled-content style="fixed-case">SCB3</jats:styled-content>, a γ‐butyrolactone from <jats:styled-content style="fixed-case"><jats:italic>S</jats:italic></jats:styled-content><jats:italic>treptomyces coelicolor</jats:italic>, and was absent from a <jats:styled-content style="fixed-case"><jats:italic>jadW123</jats:italic></jats:styled-content> mutant lacking jadomycin production. Addition of <jats:styled-content style="fixed-case">SVB1</jats:styled-content> or extract from <jats:styled-content style="fixed-case"><jats:italic>S</jats:italic></jats:styled-content><jats:italic>. coelicolor</jats:italic> to the mutant restored jadomycin production. Overall, our results revealed that the association of <jats:styled-content style="fixed-case">JadR3</jats:styled-content> and <jats:styled-content style="fixed-case">SVB1</jats:styled-content> plays an important role in controlling a regulatory mini‐network governing jadomycin biosynthesis, providing new insights into the ways in which γ‐butyrolactone/receptor systems modulate antibiotic biosynthesis in <jats:styled-content style="fixed-case"><jats:italic>S</jats:italic></jats:styled-content><jats:italic>treptomyces</jats:italic>.