The Corynebacterium glutamicum anaerobic nitrate reductase operon narKGHJI is repressed by a transcriptional regulator, ArnR, under aerobic conditions. A consensus binding site of the cAMP receptor protein (CRP)-type regulator, GlxR, was recently found upstream of the ArnR binding site in the narK promoter region. Here we investigated the involvement of GlxR and cAMP in expression of the narKGHJI operon in vivo. Electrophoretic mobility shift assays showed that the putative GlxR binding motif in the narK promoter region is essential for the cAMP-dependent binding of GlxR. Promoter-reporter assays showed that mutation in the GlxR binding site resulted in significant reduction of narK promoter activity. Furthermore, a deletion mutant of the adenylate cyclase gene cyaB, which is involved in cAMP synthesis, exhibited a decrease in both narK promoter activity and nitrate reductase activity. These results demonstrated that C. glutamicum GlxR positively regulates narKGHJI expression in a cAMP-dependent manner.