Abstract: Previous report revealed that bldD was a global transcriptional regulator, participating in morphological development and secondary metabolism in Streptomyces. Here, we studied the effects of overexpression bldD gene on spinosad biosynthesis and spore formation in Saccharopolyspora spinosa. Cm-P_ermE gene segment and bldD gene were amplified, then spliced by overlap PCR. Thus, the open reading frame of bldD was placed under the control of the promoter for erythromycin resistance gene P_ermE. The cm-P_ermE-bldD frangment was cloned into Escherichia coli-Streptomyces shuttle vector pUC-spn by Red/ET homologous recombination, generating recombinant vector pUC-spn-P_ermE-bldD. This was introduced into S. spinosa from E. coli S17 through conjugal transfer, and integrated to the chromosome of S. spinosa via homologous recombination. Apramycin resistant colonies were picked up and identified by PCR. Positive transconjugants of which bldD gene was successfully integrated into chromosome of S. spinosa SP06081 were chosen for further study. On BHI and TSB agar, the sporulation of recombination strain S. spinosa-BldD was apparently inhibited. Flask fermentation demonstrated that the recombination strain overproduced 1.35-fold spinosad compared with the control strain. The recombination strain, S. spinosa-BldD, was genetically stable. These findings revealed that overexpression of bldD in S. spinosa had a negative effect on sporulation in some cases, and effectively promoted spinosad biosynthesis in S. spinosa, which provides a basis for improving spinosad production by overexpression of other positive regulatory genes.