Production of secondary metabolites in Streptomyces is regulated by a complex regulatory network precisely, elaborately, and hierarchically. One of the main reasons for the low yields of some high-value secondary metabolites is the repressed expression of their biosynthetic gene clusters, supposedly by some gene cluster out-situated negative regulators. Identification of these repressors and removal of the inhibitory effects based on the regulatory mechanisms will be an effective way to improve their yields. For proof of the concept, using an antibiotic daptomycin from Streptomyces roseosporus, we introduced Himar1-based random mutagenesis combined with a reporter-guided screening strategy to identify a transcriptional regulator PhaR, whose loss-of-function deletion led to about 2.68-fold increase of the gene cluster expression and approximately 6.14-fold or 43% increased daptomycin production in the flask fermentation or in the fed-batch fermentation, respectively. Further study showed that PhaR negatively regulates the expression of daptomycin biosynthetic gene cluster by direct binding to its promoter (dptEp). Moreover, phaR expression gradually drops down during fermentation, and PhaR is positively auto-regulated by directly binding to its own promoter, which results in positive feedback regulation to persistently reduce phaR expression. Meanwhile, the declining PhaR protein remove its repressive effects during daptomycin production. All these results support that our strategy would be a powerful method for genetic screening and rational engineering for the yield improvement of antibiotics, and could be potentially used widely in other Streptomyces species.