Dental caries is one of the most prevalent chronic diseases worldwide, and the cariogenic biofilm formed by Streptococcus mutans plays a central role in its pathogenesis. Although bacterial membrane vesicles (MVs) are recognized as critical mediators of intercellular communication and virulence, the mechanisms regulating MVs biogenesis and their precise contribution to biofilm development remain poorly understood. Previous multi-omics analyses suggested that the ABC transporter system RcrRPQ might be involved in MVs production. TO test this hypothesis, we constructed single-gene deletion mutants of rcrR, rcrP, and rcrQ in S. mutans UA159. We systematically quantified MVs production, characterized vesicle cargo through proteomics and metabolomics, and assessed the functional impact of these vesicles on biofilm formation. Deletion of rcrR, rcrP, or rcrQ led to a significant increase in MVs release. Furthermore, MVs derived from the mutants exhibited a markedly elevated content of extracellular DNA (eDNA) and substantial remodeling of the protein cargo. Functional studies revealed a striking finding: at a low concentration (2.5 μg/mL), these MVs strongly promoted biofilm formation without increasing bacterial biomass. Instead, the enhanced biofilm formation was primarily attributed to accelerated extracellular matrix accumulation, resulting from MVs-induced upregulation of gtfB, gtfC, and gtfD and subsequent overproduction of extracellular polysaccharides (EPS). The combined enrichment of eDNA and EPS further strengthened the three-dimensional biofilm structure. In conclusion, our results demonstrate that the RcrRPQ system acts as a negative regulator of MVs biogenesis and cargo loading in S. mutans. Loss of RcrRPQ enhances MVs production, remodels vesicle cargo, and ultimately promotes biofilm maturation through matrix remodeling rather than bacterial proliferation. These findings reveal a novel RcrRPQ–MVs–biofilm matrix regulatory axis and highlight membrane vesicles as promising targets for the development of new anti-caries strategies.