Validamycin A (VAL-A) is a widely used antifungal antibiotic for the treatment of sheath blight disease of rice and other plants. It can be produced from agro-industrial by-products by Streptomyces hygroscopicus 5008. To enhance its production titer, in this work, the entire val gene cluster was amplified in tandem in S. hygroscopicus 5008 by integrating the zouA-mediated DNA amplification system into between the two boundaries of val gene cluster, resulting in multiple copies (mainly three to five) of the val gene cluster. The genetic stability of the amplified copies was confirmed by Southern blot and fermentation experiments. In shake flask fermentation, the recombinant strain (TC03) led to a 34 % enhancement of VAL-A production titer compared to that of the wild-type strain, while the accumulation of intermediate validoxylamine A was decreased in TC03. Additionally, both the structural gene transcription levels and the ValG enzyme activity were significantly increased in TC03. This work demonstrated that the amplification of the val gene cluster was an efficient strategy to enhance VAL-A production by S. hygroscopicus 5008, and the information obtained would be helpful for engineering other interesting antibiotic biosynthesis by gene cluster amplification.