Streptomyces avermitilis is well known as the producer of anthelmintic agent avermectins, which are widely used in agriculture, veterinary medicine, and human medicine. aveI encodes a TetR-family regulator, which is the homolog of AtrA. It was reported that deletion of aveI caused enhanced avermectin production. In this study, we investigated the regulatory function of the AveI in S. avermitilis. By binding to the 15-nt palindromic sequence in the promoter regions, AveI directly regulates at least 35 genes. AveI represses avermectin production by directly regulating the transcription of the cluster-situated regulator gene aveR and structural genes aveA1, aveA3, and aveD. AveI represses oligomycin production by repressing the CSR gene olmRII and structural genes olmC. AveI activates melanin biosynthesis by activating the expression of melC1C2 operon. AveI activates morphological differentiation by activating the expression of ssgR and ssgD genes, repressing the expression of wblI gene. Besides, AveI regulates many genes involved in primary metabolism, including substrates transport, the metabolism of amino acids, lipids, and carbohydrates. Therefore, AveI functions as a global regulator in S. avermitilis, controls not only secondary metabolism and morphological differentiation, but also primary metabolism.