Glucosyltransferase-C (GTFC) is a virulence factor of Streptococcus mutans. Additionally, GTFC represents an essential element required for bacterial cell coherence, allowing for the formation of dental plaque, which leads to dental caries. As such, monoclonal antibodies (MAbs) against S. mutans are believed to offer some protection against dental caries. In the current study, we amplified an approximately 1.5 kb fragment of the N-terminal half of the S. mutans gtfC gene by PCR, then induced expression of this gene. This protein was designated GTFCN. After the expressed protein was purified, it was used as an immunogen and injected into BALB/c mice. We selected and established two MAbs by producing hybridomas (HCN17 and HCN37). The anti-GTFCN antibody isotype was confirmed as IgG2a for HCN17 and IgG2b for HCN37. The anti-GTFCN antibody was found to specifically react with the GTFCN protein. The enzymatic activity of the crude glucosyltransferase of S. mutans GS-5 was significantly inhibited at a concentration of 350 ng of MAb/mL. These results suggest that the monoclonal anti-GTFCN antibodies could represent an alternative modality for passive immunization to prevent S. mutans aggregation and dental plaque.