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 OD600 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.