Abstract: Spiramycin is a 16-membered macrolide antibiotic which is produced by Streptomyces ambofaciens. It is widely used in human medicine because of its activity against bacteria. Several impurities were easily accumulated in spiramycin-fermentation broth, especially impurity D, in which the forosamine sugar is replaced by mycarose. To improve the yield of spiramycin I and reduce the content of impurity D, the effect of glycerol addition on the fermentation of Streptomyces ambofaciens was studied and the possible mechanism was analyzed. In this study, HPLC was used to determine the fermentation titer of spiramycin I and the content of impurity. The adding of glycerol with a final concentration of 0.4% at 24 h can increase the spiramycin I titer by 17.12%, and decrease the content of impurity D by 21.39% in shake flask fermentation. The results of fermentation obtanined in a 15 L bioreactor showed that the addition of glycerol at 15 h could promote spiramycin I production with a final titer of 23 314 U/mL, 27.20% higher than control (18 329 U/mL), and the final impurity D content was 8.84%, 26.27% lower than control (11.99%). The level of genes transcription related to synthesis of spiramycin I was investigated by real-time PCR. The transcription analysis showed that the polyketide synthase gene srm10 (for lactone ring synthesis), aminotransferase gene srm20 (for forosamine synthesis) and methyltransferase gene srm33 (for mycarose synthesis) were increased for almost 3−5 folds in the early stage of fermentation. This study indicates that the addition of glycerol in the early stage of fermentation can promote the transcription of genes involved in lactone ring and glycosyl synthesis, leading to an increase in the titer of spiramycin I and a decrease in content of impurity D.