Three genes, nadA, nadB, and nadC, involved in NAD de novo biosynthesis are broadly conserved in the genomes of numerous bacterial species. In the genome of Corynebacterium glutamicum, nadA and nadC but not nadB are annotated. The nadA and nadC genes are located in a gene cluster containing two other genes, designated ndnR and nadS herein. ndnR encodes a member of the Nudix-related transcriptional regulator (NrtR) family. nadS encodes a homologue of cysteine desulfurase involved in Fe-S cluster assembly. The gene cluster ndnR-nadA-nadC-nadS is genetically characterized herein. Mutant strains deficient in nadA, nadC, or nadS required exogenous nicotinate for growth, and the nicotinate auxotrophy was complemented by introduction of the corresponding gene in trans, indicating that each of these genes is essential for growth in the absence of an exogenous source of NAD biosynthesis. The results of reverse transcriptase PCR analyses and ndnR promoter-lacZ expression analyses revealed that the expression of ndnR, nadA, nadC, and nadS genes was markedly and coordinately repressed by nicotinate. The expression of these genes was enhanced by the disruption of ndnR, resulting in the loss of the nicotinate-responsive regulation of gene expression. These results suggest that NdnR acts as a transcriptional repressor of NAD de novo biosynthesis genes and plays an essential role in the regulation of the response to nicotinate.