In our previous studies, AveI was identified as a negative regulator for avermectin biosynthesis in Streptomyces avermitilis NRRL8165, and the aveI-null mutant of NRRL8165 could produce at least 10-fold more avermectin B1a than its wild-type strain. In order to explore the regulatory mechanism by which aveI affects avermectin biosynthesis, in this study, we performed a global comparative gene expression analysis between aveI deletion mutant 8165DeltaI and its wild-type strain using NimbleGen microarrays in combination with real-time reverse transcriptase-PCR. The results showed the aveI deletion has caused global changes beyond the avermectin biosynthetic gene cluster. The aveI gene not only negatively affected expression of the avermectin biosynthetic gene cluster but also affected expression of oligomycin and filipin biosynthetic clusters. In addition, the genes involved in precursor biosyntheses for avermectin or other antibiotics, such as crotonyl-CoA reductase and methylmalonyl-CoA decarboxylase, were also upregulated in aveI mutant. Furthermore, genes in several key primary metabolic pathways, such as protein synthesis and fatty acid metabolism, were found downregulated in the mutant. These results suggested that the aveI gene may be functioning as a global regulator involved in directing carbon flux from primary to secondary metabolism.