In order to investigate effects of leucyl aminopeptidase on mycelia morphology, growth rate, spinosad yield and protein expression in Saccharopolyspora spinosa by disrupting its encoding gene pepA and analyzing the characteristics of engineered S. spinosa.The pepA gene of S. spinosa was amplified based on the conserved sequence and cloned into Escherichia coli-Streptomyces shuttle vector pOJ260 to generate pOJ260- pepA, which was transformed into S. spinosa by conjugation. Mycelium observation, SDS-PAGE and HPLC were used to analyze the engineered strain.Mycelia in S. sp-ΔpepA displayed a much higher degree of fragmentation and fewer branches compared to that of parental strain. Meanwhile, the growth rate of S. sp-ΔpepA was retarded and its biomass was reduced. Shake-flask fermentation demonstrated that spinosad yield increased by 122% in S. sp-ΔpepA strain compared to that of parental strain. SDS-PAGE analysis showed that protein expression profile of the engineered strain significantly changed.The pepA gene negatively regulates the biosynthesis of spinosad and disruption of pepA gene could affect the mycelial morphology and growth of S. spinosa.