Besides metabolic pathways and regulatory networks, transport systems are also pivotal for cellular metabolism and hyper-production of biochemicals using microbial cell factories. Identification and characterization of transporters are therefore of great significance for understanding and engineering of transport reactions. Herein, a novel l-glutamate exporter MscCG2 that extensively exists in Corynebacterium glutamicum strains but is distinct from the only known l-glutamate exporter MscCG was discovered in an industrial l-glutamate producing C. glutamicum MscCG2 was predicted to possess three transmembrane helices in the N-terminal region and located in the cytoplasmic membrane, which are typical structural characteristics of the mechanosensitive channel of small conductance. MscCG2 has a low amino acid sequence identity (23%) to MscCG and evolved separately from MscCG with four transmembrane helices. Despite the considerable differences between MscCG2 and MscCG in sequence and structure, gene deletion and complementation confirmed that MscCG2 also functioned as an l-glutamate exporter and an osmotic safety valve in C. glutamicum Besides, transcriptional analysis showed that MscCG2 and MscCG genes were transcribed in similar patterns and not induced by l-glutamate producing conditions. It was also demonstrated that MscCG2-mediated l-glutamate excretion was activated by biotin limitation or penicillin treatment and constitutive l-glutamate excretion was triggered by gain-of-function mutation of MscCG2 (A151V). Discovery of MscCG2 will enrich the understanding of bacterial amino acid transport and provide additional targets for exporter engineering.IMPORTANCEExchange of matter, energy and information with surroundings is fundamental for cellular metabolism. Therefore, studying transport systems that are essential for these processes is of great significance. Besides, transport systems of bacterial cells are usually related to product excretion as well as product re-uptake, making transporter engineering a useful strategy for strain improvement. The significance of our research is in identifying and characterizing a novel l-glutamate exporter from the industrial workhorse Corynebacterium glutamicum, which will enrich the understanding of l-glutamate excretion and provide a new target for studying bacterial amino acid transport and engineering transport reactions.