[Objective] The regulatory effect of arginine regulator ArgR on the biosynthesis of exopolysaccharides (EPS) was studied in Streptococcus thermophilus. [Methods] ArgR from S. thermophilus was heterologously expressed by Escherichia coli, and purified by urea denaturation refolding and Ni²⁺ affinity chromatography. The interaction and kinetic information between ArgR and eps promoter P_epsA were detected by electrophoretic mobility shift assays (EMSA) and biolayer interferometry (BLI). The yield alteration of EPS was determined by phenol-sulfuric acid assay when the gene argR overexpressed or repressed. [Results] Heterologous expression of ArgR was formed inclusion body, and 2.95 mg/mL of soluble protein was achieved by urea denaturation refolding. EMSA and BLI analysis showed that ArgR can specifically bind with the promoter P_epsA and their affinity was high because of the low dissociation. Increased expression of argR gene reduced EPS synthesis and the suppression raised. [Conclusion] It is the first time to report that ArgR can specifically bind the promoter of eps gene cluster and negatively regulate EPS synthesis in S. thermophilus.