The effects of ammonium ion on the synthesis of biotechspiramycin were investigated. It was proved that the percentage of isovalerylspiramycin III was greatly enhanced in the medium with low concentration of ammonium ion. By greatly reducing ammonium ion concentration in the medium from 62.5mmol/L to 2.5mmol/L, the percentage of isovalerylspiramycin III increased from 5.43% to 28.59% . However, the titer of biotechspiramycin decreased to 107microg/mL in the medium with low concentration of ammonium ion, which was 14.4% lower than that in the medium with high concentration of ammonium ion, due to lack of nitrogen source. The results also showed that the activity of valine dehydrogenase, the key enzyme of leucine catabolic pathway, in the fermentation with high concentration of ammonium ion was lower than that with low ammonium ion concentration. This would lead to the limitation of isovaleryl-CoA, which is the substrate of isovaleryl transfer reaction, and then the decline of percentage of isovalerylspiramycin. The percentage of isovalerylspiramycin III further increased to 37.84% by addition of 0.3mg/mL leucine to the fermentation broth at 36h cultivation.