Avermectin and its analogs are major commercial antiparasitic agents in the fields of animal health, agriculture, and human infections. Previously, comparative transcriptome analysis between the low-producer S. avermitilis ATCC31267 and the high-producer S. avermitilis ATCC31780 using a S. avermitilis whole genome chip revealed that 50 genes were overexpressed at least two-fold higher in S. avermitilis ATCC31780. To verify the biological significance of some of the transcriptomics-guided targets, five putative regulatory genes were individually cloned under the strong-and-constitutive promoter of the Streptomyces expression vector pSE34, followed by the transformation into the low-producer S. avermitilis ATCC31267. Among the putative genes tested, three regulatory genes including SAV213, SAV3818, and SAV4023 exhibited stimulatory effects on avermectin production in S. avermitilis ATCC31267. Moreover, overexpression of SAV3818 also stimulated actinorhodin production in both S. coelicolor M145 and S. lividans TK21, implying that the SAV3818, a putative TetR-family transcriptional regulator, could be a global upregulator acting in antibiotic production in Streptomyces species.