Streptococcus mutans, a normal inhabitant of dental plaque, is considered a primary etiological agent of dental caries. Its main virulence factors are acidogenicity and aciduricity, the abilities to produce acid and to survive and grow at low pH, respectively. Metabolic processes are finely regulated following acid exposure in S. mutans. Proteome analysis of S. mutans demonstrated that lactoylglutathione lyase (LGL) was up-regulated during acid challenge. The LGL enzyme catalyzes the conversion of toxic methylglyoxal, derived from glycolysis, to S-D-lactoylglutathione. Methylglyoxal inhibits the growth of cells in all types of organisms. The current study aimed to investigate the relationship between LGL and aciduricity and acidogenicity in S. mutans. An S. mutans isogenic mutant defective in lgl (LGLKO) was created, and its growth kinetics were characterized. Insertional inactivation of lgl resulted in an acid-sensitive phenotype. However, the glycolytic rate at pH 5.0 was greater for LGLKO than for S. mutans UA159 wild-type cells. LGL was involved in the detoxification of methylglyoxal, illustrated by the absence of enzyme activity in LGLKO and the hypersensitivity of LGLKO to methylglyoxal, compared with UA159 (MIC of 3.9 and 15.6 mM, respectively). Transcriptional analysis of lgl conducted by quantitative real-time PCR revealed that lgl was up-regulated (approximately sevenfold) during the exponential growth phase compared with that in the stationary growth phase. Gene expression studies conducted at low pH demonstrated that lgl was induced during acidic growth (approximately 3.5-fold) and following acid adaptation (approximately 2-fold). This study demonstrates that in S. mutans, LGL functions in the detoxification of methylglyoxal, resulting in increased aciduricity.