Ansamitocins isolated from Actinosynnema pretiosum, potent antitumor compounds, belong to the family of maytansinoids, and the antibody-maytansinoid conjugates are currently under different phases of clinical trials. The clinical applications of ansamitocins have stimulated extensive studies to improve their production yields. In this study, we investigated the function of a pathway-specific Emphasis Type=S treptomyces antibiotic regulatory protein (SARP) family regulator, Asm18, and observed that ectopic overexpression of the asm18 gene increased the production of N-demethyl-4,5-desepoxy-maytansinol (2) to 50 mg/L in the HGF052 + pJTU824-asm18 strain, an increase by 4.7-fold compared to that of the control strain HGF052 + pJTU824. Real-time PCR analysis showed that the overexpression of the asm18 gene selectively increased the transcription levels of the genes involved in the biosynthesis of the starter unit (asm43), polyketide assembly (asmA), post-PKS modification (asm21), as well as the transcription levels of the regulatory gene (asm8), which is a specific LAL-type activator in ansamitocin biosynthesis. With the increase of fermentation titre, seven ansamitocin analogs (1-7) including three new ones (1, 5, and 6) and maytansinol (7) were isolated from the HGF052 + pJTU824-asm18 strain. Our results not only pave the way for further improving the production of ansamitocin analogs but also indicate that the post-PKS modifications of ansamitocin biosynthesis are flexible, which brings a potential of producing maytansinol, the most fascinating intermediate for the synthesis of antibody-maytansinoid conjugates, by optimizing the HGF052 and/or HGF052 + pJTU824-asm18 strains.