Glucosyltransferases GtfB, GtfC, and GtfD of Streptococcus mutans are virulent factors involved in dental caries. Consequently, they are considered to be target molecules in the development of vaccines against dental caries. Among them, GtfD plays a significant role in the sucrose-dependent cellular adhesion of S. mutans, and a number of studies have suggested that the N-terminus of GtfD is an important part of its role in enzymatic activity. In this study, we generated monoclonal antibodies against the N-terminus of GtfD (anti-GtfDN antibody) in an initial attempt to investigate its preventive efficacy against dental caries. To obtain anti-GtfDN monoclonal antibodies, the gene for the N-terminus of gtfD (2 kb) was cloned into an Escherichia coli expression vector, pQE30; then the expressed protein (about 75 kDa) was purified. The purified GtfDN protein was injected into BALB/c mice, and hybridoma clones were established. We obtained three hybridoma clones (HDN9, HDN11, and HDN28) capable of producing anti-GtfDN antibodies. Their binding specificity was characterized by ELISA, dot blot, and Western blot analysis after purification using affinity column chromatography. The isotype of the monoclonal antibodies was confirmed to be IgG2a.