Streptococcus mutans in dental biofilms often faces life-threatening threats such as killing by antimicrobial molecules from competing species or from the host. The ability of S. mutans to cope with such threats is crucial for its survival and persistence in dental biofilms. By screening a transposon mutant library, we identified 11 transposon insertion mutants that were sensitive to bacitracin. Two of these mutants, XTn-01 and XTn-03, had an independent insertion in the same locus, SMU.244, which encoded a homologue of undecaprenyl pyrophosphate phosphatase (UppP). In this study, we describe the genetic and phenotypic characterization of SMU.244 in antibiotic resistance. The results revealed that deletion of SMU.244 results in a mutant (XTΔ244) that is highly sensitive to bacitracin, but confers more resistance to lactococcin G, a class IIb bacteriocin. Introduction of the intact SMU.244 into XTΔ244 in trans completely restores its resistance to bacitracin and the susceptibility to lactococcin G. The XTΔ244 was also defective in forming the WT biofilm, although its growth was not significantly affected. Using recombinant protein technology, we demonstrated that the SMU.244-encoded protein displays enzyme activity to catalyse dephosphorylation of the substrate. The lux transcriptional reporter assays showed that S. mutans maintains a moderate level of expression of SMU.244 in the absence of bacitracin, but bacitracin at sub-MICs can further induce its expression. We concluded that SMU.244 encodes an UppP protein that plays important roles in cell wall biosynthesis and bacitracin resistance in S. mutans. The results described here may further our understanding of the molecular mechanisms by which S. mutans copes with antibiotics such as bacitracin.