Streptococcus mutans (S. mutans) is a primary oral cariogenic bacterium. The OpuB transporter regulates osmotic pressure in Bacillus subtilis; however, its role in S. mutans remains unexplored. Our earlier research indicated that, under acid stress, the OpuB ABC-transport pathway in S. mutans membrane vesicles undergoes significant changes, implying its critical role in the bacterium's response to environmental stress. In this study, we constructed an opuB-deficient strain (Smu_opuB) and compared it with the wild-type strain. The results revealed that knocking out opuB enhanced the survival of planktonic S. mutans in an acidic environment, increased extracellular polysaccharide and biofilm production under acid stress, altered biofilm structure, and upregulated the expression of related virulence factors. These findings imply that opuB is instrumental in regulating acid resistance and biofilm formation in S. mutans, thereby conferring a survival advantage. This study provides compelling evidence of opuB being pivotal in S. mutans' acid resistance and biofilm formation, deepening our understanding of its functional mechanisms and establishing a foundation for future research on its role in S. mutans.