To identify useful native promoters of Corynebacterium glutamicum for fine-tuning of gene expression in metabolic engineering. Sixteen native promoters of C. glutamicum were characterized. These promoters covered a strength range of 31-fold with small increments and exhibited relatively stable activity during the whole growth phase using beta-galactosidase as the reporter. The mRNA level and enzymatic activity of the lacZ reporter gene exhibited high correlation (R (2) = 0.96) under the control of these promoters. Sequence analysis found that strong promoters had high similarity of the -10 hexamer to the consensus sequence and preference of the AT-rich UP element upstream the -35 region. To test the utility of the promoter library, the characterized native promoters were applied to modulate the sucCD-encoded succinyl-CoA synthetase expression for l-lysine overproduction. The native promoters with various strengths realize the efficient and precise regulation of gene expression in metabolic engineering of C. glutamicum.