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The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits potent selective activity against pathogenic protozoa and fungi. It is one of the important effective components in Agricultural Antibiotic120, which has been widely used as naturally-originated agents for treatment of crop decay in China. The chemical synthesis of anisomycin has recently been reported, but the complex process with low productivity made the biosynthesis still to be a vital mainstay in efforts. The biosynthetic gene cluster (BGC) of anisomycin in Streptomyces hygrospinosus var. beijingensis has been identified in our previous work, while poor understanding of the regulatory mechanism limited the yield enhancement via regulation engineering of S. hygrospinosus var. beijingensis. In this study here, we characterized AniF as an indispensable LuxR family transcriptional regulator for the activation of anisomycin biosynthesis. The genetic manipulations of aniF and the real-time quantitative PCR (RT-qPCR) revealed that it positively regulated the transcription of the anisomycin BGC. Moreover, the overexpression of aniF contributed to the improvement of the production of anisomycin and its derivatives. Dissection of the mechanism underlying the function of AniF revealed that it directly activated the transcription of the genes aniR-G involved in anisomycin biosynthesis. Especially, one AniF-binding site in the promoter region of aniR was identified by DNase I footprinting assay and an inverted repeat sequence (5′-GGGC-3′) composed of two 4-nt half sites in the protected region was found. Taken together, our systematic study confirmed the positive regulatory role of AniF and might facilitate the future construction of engineering strains with high productivity of anisomycin and its derivatives. Keywords: Anisomycin, Agricultural Antibiotic120, Biosynthesis, AniF, LuxR regulator, Streptomyces