Ansamitocin P-3 (AP-3), an amacrocyclic lactam compound, is produced by Actinosynnema pretiosum. As a group of maytansinoid antibiotics, ansamitocins have an extraordinary antitumor activity by blocking the assembly of tubulin forming into functional microtubules. The biosynthesis of ansamitocins is initialized by the formation of UDP-glucose (UDPG) which is converted from glucose-1-phosphate (G1P). In this study, we focused on the influence of enhancement of UDPG biosynthesis on the production of ansamitocins in A. pretiosum. The homologous overexpressions of phosphoglucomutase, starch phosphorylase, and UTP-G1P uridylyltransferase, respectively, could largely increase the pool sizes of G1P and UDPG and result in improved AP-3 production. The elevated intracellular glucose-6-phosphate (G6P) level provided by the enhanced glyconeogenesis had, however, no significant effects on the biosynthesis of AP-3. The G6P-G1P-UDPG pathway was therefore systematically engineered by multiple genetic modifications, and a significant increase in AP-3 production was achieved (168 mg/L of AP-3 in flask culture, 40 % higher than the control strain). We also found that the enhancement of starch assimilation pathway could also improve the assembly of AP-3 to some extent. In addition, heterologous gene overexpression from Actinosynnema mirum could result in more AP-3 biosynthesis in comparison to the corresponding homologous overexpression, suggesting an alternative and promising avenue of metabolic engineering strategy for improving AP-3 production.