Dental caries is a common and multifactorial biofilm disease that is associated with dietary habits and microbiota. Among the various pathogens inducing caries, S. mutans is the most extensively studied. Promoting oral health with probiotics has gained considerable attention. Lactobacillus paracasei (L. paracasei) strains were reported to modulate the gut microbiota and enhance host resistance to disease. Our previous research has found that L. paracasei ET-22 (ET-22) could inhibit S. mutans biofilms in vitro. However, the preventive effect in vivo and functional mechanism of ET-22 on dental caries were unclear. In this study, the preventive effects of ET-22 on dental caries in mice were checked. Meanwhile, the functional mechanism of ET-22 was further investigated. Results showed that the supplementation of ET-22 in drinking water significantly improved the caries scoring of mice. The microbiota of dental plaques revealed that the live and heat-killed ET-22 similarly regulated the microbial structure in plaque biofilms. Functional prediction of PICRUSt showed that the addition of live and heat-killed ET-22 may inhibit biofilm formation. By the in vitro trials, the live and heat-killed ET-22 indeed inhibited the construction of S. mutans biofilms and EPS productions of biofilms. This evidence suggests that ET-22 can restrain dental caries by regulating the microbiota of dental plaques and inhibiting biofilm formation, which may be partly mediated by the body components of ET-22.