The halophilic lactic acid bacteriumTetragenococcus halophilushas been widely used in high-salinity fermentation processes of food. Previous studies have indicated that the catabolism of arginine may contribute to the osmotic stress adaptation ofT. halophilus. Unusually, in the chromosome ofT. halophilus, preceding the arginine deiminase (ADI) operon, locate two co-transcribed genes, both encoding an ArgR regulator; similar structure was rarely found and the roles of the regulators have not been demonstrated. In the current study, regulatory roles of these two nonidentical ArgR regulators on the arginine metabolism ofT. halophiluswere investigated. The results show that these two regulators play different roles in arginine metabolism, ArgR1 acts as a negative regulator of the ADI pathway by binding to the promoter sequences and repressing the transcription of genes, and the addition of arginine or hyper-osmotic stress conditions can abolish the ArgR1 repression, whereas ArgR2 negatively regulates the genes involved in arginine biosynthesis. Our study found that despite the commonly known roles of the ArgR regulators as the activator of arginine catabolism and the repressor of arginine biosynthesis, which are found in most studied bacteria possessed one ArgR regulator, the two nonidentical ArgR regulators ofT. halophilusboth act as repressors, and the repression by which is regulated when sensing changes of environments. By revealing the regulation of arginine metabolism, the current study provides molecular insights and potential tools for future applications of halophiles in biotechnology.