Ansamitocin P-3 (AP-3), a 19-membered polyketide macrocyclic lactam, has potent antitumor activity. Our previous study showed that a relatively low organic nitrogen concentration in culture medium could significantly improve AP-3 production of Actinosynnema pretiosum. In the present study, we aimed to reveal the possible reasons for this improvement through metabolomic and gene transcriptional analytical methods. At the same time, a metabolic pathway profile based on metabolome data and pathway correlation information was performed to obtain a systematic view of the metabolic network modulations of A. pretiosum. Orthogonal partial least squares discriminant analysis showed that nine and eleven key metabolites directly associated with AP-3 production at growth phase and ansamitocin production phase, respectively. In-depth pathway analysis results highlighted that low organic nitrogen availability had significant impacts on central carbon metabolism and amino acid metabolic pathways of A. pretiosum and these metabolic responses were found to be beneficial to precursor supply and ansamitocin biosynthesis. Furthermore, real-time PCR results showed that the transcription of genes involved in precursor and ansamitocin biosynthetic pathways were remarkably upregulated under low organic nitrogen condition thus directing increased carbon flux toward ansamitocin biosynthesis. More importantly, the metabolic pathway analysis demonstrated a competitive relationship between fatty acid and AP-3 biosynthesis could significantly affect the accumulation of AP-3. Our findings provided new knowledge on the organic nitrogen metabolism and ansamitocin biosynthetic precursor in A. pretiosum and identified several important rate-limiting steps involved in ansamitocin biosynthesis thus providing a theoretical basis of further improvement in AP-3 production.