An industrial mutant of Streptomyces avermitilis produced avermectin at a high level in industrial complex culture medium. However, almost no avermectin was detected in the cultures of tryptone soya broth (TSB). Its wild-type strain could not synthesize avermectin. To elucidate the regulatory mechanism about avermectin production, proteomic analysis of S. avermitilis was carried out. Results showed that during avermectin biosynthesis, fatty acid metabolism and TCA cycle were repressed. Partial enrichment of glycolytic pathway indicated the critical role of glucose catabolism during avermectin biosynthesis. Some enriched enzymes in amino acid metabolic pathways (glnA, leuC) confirmed the leucine as the possible precursor of avermectin. Highly expressed stress or stress-related proteins indicated a global regulation mechanism at the onset of avermectin production. And highly expressed morphology control proteins revealed an association between hyphal morphology and avermectin production. Further, this study proofed strengthened capability to utilize carbon and nitrogen source in the industrial strain. Some stress or stress-related proteins (eshA, clpC, dnaK and grpE) expressed at low level in the industrial strain cultivated in non-production medium (lower than that in the wild-type strain), but these highly expressed at the onset of avermectin production. More sensible response to environmental stress may be responsible for it.