Corynebacterium crenatum SYA is an aerobic, Gram-positive, non-sporulating coryneform bacterium, and the mutant C. crenatum strain SYPA 5-5 can produce 30 g/l L-arginine under optimal culture conditions. In this study, the evolution of the cluster argCJBDFRGH (argC similar to H) involved in arginine biosynthesis in C. crenatum SYA, and SYPA 5-5 was investigated. Compared to the argR of its wild type C. crenatum SYA, a nucleotide substitution (C -> T) within the argR gene of the mutant C. crenatum strain SYPA 5-5 was found. The inactivation of ARGR resulted in increased enzyme activities involved in L-arginine biosynthesis and increased L-arginine production in C. crenatum. In contrast, constructing an overexpressing argR C. crenatum/pTR, a complete and functional ARGR decreased the expression of enzymes, depressed transcriptional level of the argC similar to H cluster, and reduced the production of L-arginine in C. crenatum. It was thus evident that the inactivation of an ARGR suppressor could relieve a bottleneck in downstream steps of the L-arginine biosynthetic pathway, providing a good strategy for improving L-arginine production.