Arginine utilization in P. aeruginosa as the source of carbon, nitrogen, and energy is controlled by a complicated regulatory mechanism. While ArgR in the presence of exogenous arginine is required for auto-induction of the aotJQMOP-argR operon for arginine uptake and regulation, this operon is subjected to carbon catabolite repression via an unknown mechanism. This study demonstrated that succinate exerted a stronger repression effect than glucose or pyruvate on arginine induction of an aotJ::lacZ fusion in wild type PAO1. Expression of the aotJ::lacZ fusion was analyzed against three different backgrounds, cbrAB, crc, and vfr, that have been suggested to play a role in carbon catabolite repression. These mutations exerted a negative effect on the arginine-responsive induction to different extents, with the order of cbrAB > vfr > crc. A series of aotJ::lacZ fusions with deletions in the aotJ regulatory region were constructed and the effect of exogenous arginine examined in the argR mutant and its parent strain. The results indicated that a 250-bp sequence upstream of the ArgR operator is required for the optimal induction of the operon by exogenous arginine, and revealed the presence of a cryptic promoter (P0) in this region. Electromobility shift assays with crude cell-free extracts of PAO1 revealed that a DNA-binding protein other than ArgR binds to the aforementioned 250-bp region. Through reverse genetics, two regulatory proteins MvaT and MvaU were identified and specifically interacted with the aotJ-argR regulatory region. The MvaT/MvaU are involved in the regulation of the P0 promoter. The importance of MvaT and MvaU for bacterial growth was supported by the notion that no true mvaT mvaU double knockout mutant can be constructed. This is the first case to characterize the growth phenotypes of quasi-mvaT mvaU double mutants complemented with fusions for arginine-inducible expression of mvaT or mvaU. Further analysis of the physiological significance of MvaT and MvaU revealed their involvement in swarming response and pyocyanin synthesis. The defect in pyocyanin synthesis was correlated to the diminished level of PQS, an important chemical signal compound in the quorum sensing network of P. aeruginosa.