In this research, the effects of different carbon sources on the strain growth and butenyl-spinosyn biosynthesis of Saccharopolyspora pogona were studied. The study was aimed to improve the butenyl-spinosyn biosynthesis by looking for dominant carbon sources and optimizing the fermentation medium formula. A total of eleven processing groups and one control group were set up in this experiment. The strain OD600 value and butenyl-spinosyn production in different processing groups were compared by single factor experiment for screening the optimal carbon source and fermentation medium formulation. The results showed that Saccharopolyspora pogona could grow in 9 kinds of carbon sources except for xylose and soluble starch, and exhibited good utilization rate for different types of carbon sources. Saccharopolyspora pogona has a good growth rate when using galactose, glucose, fructose and mannose as carbon sources, but just mannose can promote butenyl-spinosyn biosynthesis. The 5 g/L mannose was selected as the optimal concentration and it was found that Saccharopolyspora pogona strain concentration and butenyl-spinosyn production were 1.32 times and 1.78 times of the initial conditions, respectively. Its production significantly increased. The study provides a scientific bases and a new technical approach for the influencing mechanism study of medium carbon sources on the butenyl-spinosyn biosynthesis and the large-scale industrial production of butenyl-spinosyn.