The effects of glucose and inorganic phosphate on mycelium growth and spinosad production with Saccharopolyspora spinosa were studied. The results showed that the increase of glucose concentration from 18.6g(.)L(-1) to 58.8g(.)L(-1) could promote the mycelium growth and spinosad production. And when the glucose concentration increased from 58.8g(.)L(-1) to 79.6g(.)L(-1), no obvious change was detected but a slight drop in spinosad production was observed, whereas, when the glucose concentration increased from 79.6g(.)L(-1) to 115.3g(.)L(-1), substantial decrease in both mycelium growth and spinosad production occurred. The increase of phosphate concentration from 3.68mmol(.)L(-1) to 29.41mmol(.)L(-1) rendered corresponding increase in mycelium growth and spinosad production. When phosphate concentration increased from 29.41mmol(.)L(-1) to 44.12mmol(.)L(-1), mycelium growth slightly increased and spinosad production dropped, while when phosphate concentration increased from 44.12mmol(.)L(-1) to 57.62mmol(.)L(-1), both mycelium growth and spinosad production decreased sharply. Conclusively, the optimal initial concentration of glucose and phosphate is 58.8g(.)L(-1) and 29.41mmol(.)L(-1), respectively. The spinosad fermentation in the production medium containing 58.8g(.)L(-1) glucose and 29.41mmol(.)L(-1) phosphate was scaled up in 5-L fermentation and the spinosad production reached 507mg(.)L(-1), which was 28% higher than that in the flask fermentation.