INTRODUCTION: Dental caries is a non-communicable disease caused by dysbiosis of dental biofilm. S. mutans is considered the major pathogen. The orphan response regulator GcrR negatively regulates exopolysaccharides (EPSs) synthesis in S. mutans. We aimed to investigate the effect of GcrR on the cariogenicity of oral biofilms. A multispecies biofilm model was constructed, including S. mutans, S. sanguinis, and S. gordonii. METHODS: The morphology of multispecies biofilms was detected through scanning electron microscopy (SEM), and the structure was observed using confocal laser scanning microscopy (CLSM). The microbial composition was measured by fluorescence in situ hybridization (FISH) and qPCR. The expression of genes was detected by quantitative RT-PCR (qRT-PCR). A specific pathogen-free (SPF) rat model was used to assess the cariogenicity of the multispecies biofilms. RESULTS: The architecture of the biofilm was significantly impaired when gcrR-overexpressed S. mutans incubated with S. sanguinis, and S. gordonii (SmugcrR++S.s+S.g). The biofilm exhibited a decrease in the production of water-insoluble glucans (WIGs) and water-soluble glucans (WSGs), consistent with a decreased expression of EPS synthesis-related genes. The SmugcrR++S.s+S.g biofilm exhibited an increase in non-cariogenic species with lower lactic acid production. Furthermore, SmugcrR++S.s+S.g biofilm exhibited reduced cariogenicity. CONCLUSION: The biofilm cariogeneicity could be shifted to a less cariogenic state by increased expression of the GcrR regulator.