Ansamitocin P-3 (AP-3) is an antitumor agent produced by Actinosynnema pretiosum with a market demand for large and cheap supply, but its productivity is yet low. This work investigated the effects of ammonium in medium on the productivity, enzyme activities, and gene transcription for AP-3 biosynthesis. As observed, AP-3 production was depressed by medium ammonium, although the dry cell weight and the consumption of total sugar and isobutanol were not influenced obviously. From the onset of AP-3 accumulation, isobutyrate accumulation showed a different behavior in medium with or without ammonium. Isobutanol dehydrogenase activity was enhanced during production phase in medium with ammonium, but valine dehydrogenase activity was not substantially changed. qRT-PCR analysis revealed that transcriptional levels of structure genes asm14, asm24, asm43, and asm19 were down-regulated by medium ammonium. The transcription of regulatory gene asm2, asm29, and asm31 were slightly up-regulated while that of asm39 was down-regulated by ammonium. The results indicated that inhibition of AP-3 production by ammonium might be related to the AP-3 ester side chain supply and the repression of gene transcription responsible for 3-amino-5-hydroxybenzoic acid and methoxymalonyl-ACP biosynthesis. The information is useful for future AP-3 productivity enhancement.