Abstract: Adding glycerol made for spiramycin biosynthesis during the fermentation process of Streptomyces spiramyceticus. It increased the titer about 20%—30% more than the control that added glycerol at 48 h in shaker flasks. Moreover, the amplificatory exercises in 50 L fermentors successfully verified the stimulative effect of glycerol. And its mechanism was investigated in biosynthesis process by measuring a variety of relative organic acid content, the biomass, the concentrations of sugar and NH2-N in broth. The mechanism was that glycerol mainly increased the metabolism flux of EMP pathway and TCA cycle, could activate mycelia in the early of fermentation, and promoted the cells primary metabolism. To some extent, the yield and productivity of spiramycin were increased.