Abstract: Geldanamycin, a bioactive compound produced by Streptomyces autolyticus, possesses potent antitumor activity. However, the specific regulatory mechanism of the biosynthesis of geldanamycin is yet unclear. To further illustrate the regulatory mechanism of geldanamycin biosynthesis in S. autolyticus, the function and regulatory mechanism of Orf17, a putative MarR family regulator were studied. The HPLC analysis of the fermentation products of the orf17 mutant showed that the production of geldanamycin in the orf17 mutant decreased when compared to that in the wild-type strains, suggesting that orf17 gene might play a positive role in the biosynthesis of geldanamycin. RT-PCR analysis revealed that the expression of genes almAⅠ, almF, almM, almH, almRⅠ, almRⅡ, and almRⅢ that are involved in the geldanamycin biosynthesis decreased in orf17 mutant, implying the expression of these genes was regulated by Orf17, directly or indirectly. Furthermore, three compounds with increased production in the mutant were purified and identified as elaiophylin and its analogues. In summary, Orf17 played a positive role in regulating the biosynthesis of geldanamycin, and a negative role in the biosynthesis of elaiophylin.