AbstractSpinosad, a combination of spinosyn A and D produced bySaccharopolyspora spinosa, is a highly efficient pesticide. There has been a considerable interest in the improvement of spinosad production because of a low yield achieved by wild‐typeS. spinosa. In this study, we designed and constructed a pIBR‐SPN vector. pIBR‐SPN is an integrative vector that can be used to introduce foreign genes into the chromosome ofS. spinosa. Different combinations of genes encoding forasamine and rhamnose were synthesized and used for the construction of different recombinant plasmids. The following recombinant strains were developed:S. spinosapIBR‐SPN (only the vector),S. spinosapIBR‐SPN F (forosamine genes),S. spinosapIBR‐SPN R (rhamnose genes),S. spinosapIBR‐SPN FR (forosamine and rhamnose genes),S. spinosapIBR‐SPN FRS (forosamine, rhamnose, and SAM [S‐adenosyl‐L‐methionine synthetase] genes), andS. spinosaMUV pIBR‐SPN FR. Among these recombinant strains,S. spinosapIBR‐SPN FR produced 1394 ± 163 mg/L spinosad, which was 13‐fold higher than the wild‐type.S. spinosaMUV pIBR‐SPN FR produced 1897 (±129) mg/L spinosad, which was seven‐fold higher thanS. spinosaMUV and 17‐fold higher than the wild‐type strain.