Streptococcus mutans is an important factor in the etiology and pathogenesis of dental caries, largely owing to its ability to form a stable biofilm. Previous animal studies have indicated that rnc could decrease the amount of sulcal caries, and that the downregulation of cariogenicity might be due to its capacity to disrupt biofilm formation. However, the biofunctions by which rnc is involved in biofilm formation remain to be elucidated. In this study, we further investigate the role of rnc based on the study of mature biofilm. Scanning electron microscopy and the crystal violet assay were used to detect the biofilm forming ability. The production and distribution of exopolysaccharides within biofilm was analyzed by exopolysaccharide staining. Gel permeation chromatography was used to perform molecular weight assessment. Its adhesion force was measured by atomic force microscopy. The expression of biofilm formation-associated genes was analyzed at the mRNA level by qPCR. Here, we found that rnc could occur and function in biofilm formation by assembling well-structured, exopolysaccharide-encased, stable biofilms in S. mutans. The weakened biofilm forming ability of rnc-deficient strains was associated with the reduction of exopolysaccharide production and bacterial adhesion. Over all, these data illustrate an interesting situation in which an unappreciated regulatory gene acquired for virulence, rnc, most likely has been coopted as a potential regulator of biofilm formation in S. mutans. Further characterization of rnc may lead to the identification of a possible pathogenic biofilm-specific treatment for dental caries.