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Bacterial cells are surrounded by a peptidoglycan (PG) cell wall, which is essential for cell integrity and intrinsic biogenesis pathways; hence, the cell wall is a potential target for several antibiotics. Among several lytic transglycosylases (LTs), the mltG gene plays a crucial role in the synthesis of peripheral PG. It localises the re-modelled PGs for septum formation and cleavage across the bacterial cell wall during daughter cells separation. However, the role of mltG gene in bacterial virulence, particularly in Gram-positive bacteria during dentine biofilm and caries development, has remained unexplored. Hence, we exploited Gram-positive Streptococcus mutans cells for the very first time to construct a mltG knock-out bacterial strain, e.g., ΔmltG S. mutans. Systematic comparative investigations revealed that doubling time (Td), survival, enzymatic efficiencies, pH tolerance, bio-synthesise of lipid, proteins and DNA, biofilm formation and dentine lesions were significantly (p < 0.001) compromised in case of ΔmltG S. mutans than wild type strain. The qRT-PCR based gene expression profiling revealed that transcriptional expression of critically important genes involved in biofilm, metabolism, and stress response were dysregulated in the mutant. Besides, an incredible reduction in dentine caries development was found in the molar teeth of Wistar rats and also in human extracted teeth. Concisely, these trends obtained evidently advocated the fact that the deletion of mltG gene can be a potential target to impair the S. mutans virulence through severe growth retardation, thereby reducing the virulence potential of S. mutans., (© 2023. The Author(s).)