Butenyl-spinosyn, a highly effective biological insecticide, is produced by . However, its application has been severely hampered by its low yield. Recent studies have shown that PhoU plays a pivotal role in regulating cell growth, secondary metabolite biosynthesis and intracellular phosphate levels. Nevertheless, the function of PhoU remains ambiguous in . In this study, we investigated the effects of PhoU on the growth and the butenyl-spinosyn biosynthesis of by constructing the mutants. Overexpression of increased the production of butenyl-spinosyn to 2.2-fold that of the wild-type strain. However, the deletion resulted in a severe imbalance of intracellular phosphate levels, and suppression of the growth and butenyl-spinosyn biosynthesis. Quantitative Real-time PCR (qRT-PCR) analysis, distinctive protein detection and mass spectrometry revealed that PhoU widely regulated primary metabolism, energy metabolism and DNA repair, which implied that PhoU influences the growth and butenyl-spinosyn biosynthesis of as a global regulator.