Streptococcus agalactiae infectious disease outbreaks have occurred continuously in Oreochromis spp. farms in China since 2009. In order to further explore the regulation mechanism of S. agalactiae virulence, the function of a putative two-component system (TCS) RscSR was investigated in a highly virulent strain GD201008-001, which was isolated from moribund cultured tilapia with meningoencephalitis in Guangdong Province of China in 2010. The 3-D structures and conserved domains of histidine kinase RscS and response regulator RscR were predicted using the I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER) and SMART (https://smart. embl.de). The rscSR deletion mutant strain ΔrscSR was constructed using homologous recombination. Bacterial tolerances to acid or oxidative stress were investigated by pre-exposing to pH 5.0 or 20 mmol/L H₂O₂. RAW264.7 macrophages were used as cell models to evaluate the bacterial anti-phagocytosis ability, intracellular survival ability and cytotoxicity. The bacterial pathogenicity was then tested in adult mice. The bioinformatics prediction showed that RscS and RscR were a typical histidine kinase and a response regulator, respectively. The stimuli sensed by RscS might be membrane-associated or occur directly at membrane interface; RscR was predicted to bind DNA to directly affect the transcription of the target gene. Compared with the wild-type and complemented strains, the resistance to acid (pH 5.0) of ΔrscSR was significantly decreased (2.83 times and 1.65 times), and furthermore, its resistance to H₂O₂ was also significantly decreased (1.93 times and 1.77 times). The ΔrscSR mutant displayed significantly decreased resistance to being ingested by macrophages (2.81 times and 1.56 times) compared to that of the wild-type and complemented strains. Moreover, at 3 h after being taken up by the macrophages, the intracellular survival rate of the ΔrscSR mutant was only approximately 44%; in contrast, it was 60% both for the wild type and complemented strains. After 6 h of phagocytosis, the intracellular survival rates of the wild type, ΔrscSR and complemented strains were decreased to about 46%, 28% and 40%, respectively. The cytotoxicity assay indicated that the relative number of lysed RAW 264.7 cells infected with the ΔrscSR mutant was significantly lower, exhibiting 1.8-fold and 2.5-fold decreases, respectively, compared to the wild-type and CΔrscSR strains. The mice infected with the wild strain all died within 19 hours, while mice infected with the ΔrscSR strain all died within 48 hours. This study suggests RscSR plays an important role in stress response, intracellular survival and virulence of S. agalactiae, which will provide useful information for further exploration of pathogenic mechanism of this bacterium.