Spinosad (spinosyns A and D) is a mixture of secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. According to the pathway and regulation of spinosad biosynthesis, a rational selection procedure with u.v. mutation was performed to obtain high spinosad producing strain. The spinosad resistant mutants, the rhamnose resistant mutants, the 2-deoxygen-D-glucose resistant mutants were selected, successively. Eventually, a strain S. spinosa 4∼6 was obtained, with its production of spinosad reaching 268 mg/L, which is increased by 121% in comparison with that of the parent strain S. spinosa 1∼5. The subculture experiments indicated that the hereditary character of high production of S. spinosa 4∼6 is stable. The spinosad fermentation with S. spinosa 4∼6 was scaled up in a 10 L fermentor, and a production of 458 mg/L was obtained, which was 71% higher than the production with shaking-flask fermentation.