Glucosyltransferase-B (GTFB) of Streptococcus mutans is considered a virulence factor because of its activity in the production of insoluble glucan, which is key to the bacterial attachment onto dental surfaces, leading to the formation of dental caries. Local passive immunization with monoclonal antibodies against GTFB is considered to be an effective way to prevent dental caries. Here we amplified a 1.3 kb fragment of the N-terminal half of the gtfB gene (193-1530) of S. mutans by PCR and expressed the truncated protein (GTFBN). The expressed, purified protein was used as an immunogen in BALB/c mice. We selected and established one hybridoma (HBN8) that was capable of producing anti-GTFBN using ELISA, dot blot, and Western blot analyses. The monoclonal anti-GTFBN antibody was purified by affinity chromatography and its isotype was confirmed as IgG2a. The anti-GTFBN antibody inhibited the enzymatic activity of crude glucosyltransferase of S. mutans GS-5 in a dose-dependent manner. These data suggest that the anti-GTFBN antibody could be used as a vaccine to prevent the aggregation of S. mutans on tooth surfaces, and thus prevent the formation of dental caries.