Abstract <p>Butenyl-spinosyn, derived from <i>Saccharopolyspora pogona</i>, is a broad-spectrum and effective bioinsecticide. However, the regulatory mechanism affecting butenyl-spinosyn synthesis has not been fully elucidated, which hindered the improvement of production. Here, a high-production strain <i>S. pogona</i> H2 was generated by Cobalt-60 γ-ray mutagenesis, which showed a 2.7-fold increase in production compared to the wild-type strain <i>S. pogona</i> ASAGF58. A comparative transcriptomic analysis between <i>S. pogona</i> ASAGF58 and H2 was performed to elucidate the high-production mechanism that more precursors and energy were used to synthesize of butenyl-spinosyn. Fortunately, a PurR family transcriptional regulator TF00350 was discovered. TF00350 overexpression strain RS00350 induced morphological differentiation and butenyl-spinosyn production, ultimately leading to a 5.5-fold increase in butenyl-spinosyn production (141.5 ± 1.03&#xa0;mg/L). Through transcriptomics analysis, most genes related to purine metabolism pathway were downregulated, and the butenyl-spinosyn biosynthesis gene was upregulated by increasing the concentration of c-di-GMP and decreasing the concentration of c-di-AMP. These results provide valuable insights for further mining key regulators and improving butenyl-spinosyn production.</p> Key points <p>• <i>A high production strain of S. pogona H2 was obtained by</i> ^<i>60</i><i>Co γ-ray mutagenesis.</i></p> <p>• <i>Positive regulator TF00350 identified by transcriptomics, increasing butenyl-spinosyn production by 5.5-fold.</i></p> <p>• <i>TF00350 regulated of butenyl-spinosyn production by second messengers.</i></p>