Biofilms are a key component in bacterial communities providing protection and contributing to infectious diseases. However, mechanisms involved in S. sanguinis biofilm formation have not been clearly elucidated. Here, we report the identification of a novel S. sanguinis TetR repressor, brpT (Biofilm Regulatory Protein TetR), involved in biofilm formation. Deletion of brpT resulted in a significant increase in biofilm formation. Interestingly, the mutant accumulated more water soluble and water insoluble glucans in its biofilm compared to the wild-type and the complemented mutant. The brpT mutation led to an altered biofilm morphology and structure exhibiting a rougher appearance, uneven distribution with more filaments bound to the chains. RNA-sequencing revealed that gtfP, the only glucosyltransferase present in S. sanguinis, was significantly up-regulated. In agreement with these findings, we independently observed that deletion of gtfP in S. sanguinis led to reduced biofilm and low levels of water soluble and insoluble glucans. These results suggest that brpT is involved in the regulation of the gtfP-mediated exopolysaccharide synthesis and controls S. sanguinis biofilm formation. The deletion of brpT may have a potential therapeutic application in regulating S. sanguinis colonization in the oral cavity and the prevention of dental caries.