Tunicamycin is a nucleoside type antibiotic with a potent antibacterial activity. Tunicamycin gene cluster in Streptomyces clavuligerus lacks a cluster-situated regulator (CSR). Therefore, there is no information about its regulation in the cell. To have an insight about the regulation of tunicamycin biosynthesis, the possible effects of BldG pleiotropic regulator involved in the control of secondary metabolite production in S. clavuligerus were investigated. To overexpress bldG in the cell, strains containing multiple copies of the gene expressed from PglpF promoter of S. clavuligerus pLB1, and an additional bldG integrated in the chromosome of S. clavuligerus pLB2, were constructed. S. clavuligerus pLB1 and S. clavuligerus pLB2 fermentations resulted in 16.4- and 13.8-fold higher specific tunicamycin titers, respectively, in comparison to wild type by confirming quantitative reverse-transcription PCR (qRT-PCR) data. However, qRT-PCR expression analysis of tunicamycin genes in S. clavuligerus ΔbldG constructed by Bignell with coworkers [1] showed that gene expressions at T36 (except for SCLAV_4274 and SCLAV_4275) were from 3.6- to 57.9-fold reduced compared to wild type. The tunicamycin titers were lower in S. clavuligerus ΔbldG than in wild type, as well. Consequently, the data presented here is the first report indicating a positive role of BldG on tunicamycin.