Abstract: Ansamitocins are a group of macrolide antibiotics, which include five components, i.e., AP 1, AP 2, AP 3, AP 3′ and AP 4. Among these components, AP 3 is an active and potent antitumor maytansinoid, which is also the target fermentation product. The yield and composition of ansamitocins by Actinosynnema mirum were observed to be significantly affected by the addition of branched chain amino acids (BCCAs). Experiment results showed that the AP 3 yield has doubled with the supplementation of 0.5 g/L valine at 48 h of fermentation, while adding 0.5 g/L leucine or isoleucine exhibited significant inhibition on AP 3 yield (decreased by 58.8% or 83.3%, respectively). Instead, the contents of AP 2 and AP 4 were increased when leucine or isoleucine was added into the medium. In addition, the secretion of succinic acid and acetic acid was reduced with the addition of valine but increased by isoleucine, implying that the metabolism of BCCAs in Actinosynnema. mirum influences not only the synthesis of polyketone and side chains, but also the metabolic state of cells.