Corynebacterium glutamicum is an industrially important producer of amino acids and an emerging model system for the Corynebacterineae. The glxR gene of C. glutamicum ATCC 13032 encodes a DNA binding transcription factor of the Crp-Fnr protein family. Available data indicated a prominent role of GlxR in the transcriptional regulatory network of C. glutamicum. We have used recently published whole-genome annotations of several corynebacteria to derive further evidence for GlxR-mediated transcriptional regulation from a comparative genomics approach. A bioinformatic strategy detected 22 genes belonging to a proposed GlxR core regulon in corynebacteria. Binding of purified GlxR protein to 40-mer oligonucleotides representing predicted GlxR binding sites in the upstream region of core genes from C. glutamicum ATCC 13032 was verified in vitro by electrophoretic mobility shift assays. Based on current data, a reliable genome-scale prediction of GlxR binding sites was performed, indicating that about 14% of the annotated C. glutamicum genes might be under direct transcriptional control by GlxR. Integration of GlxR-mediated regulatory interactions with the data stored in CoryneRegNet enabled the reconstruction of a refined transcriptional regulatory network model for C. glutamicum. This network model exhibited a hierarchical and modular structure and is characterized by the presence of master regulators in each functional module, with GlxR serving as the dominating regulatory hub.