Abstract: This study aimed to investigate the impact of probiotic-enhanced children's formula milk on oral health and the oral microbiome in preschool children. A randomized controlled trial was conducted with 163 preschool children, including 93 in the intervention group and 70 in the control group. The intervention group consumed formula milk containing Bifidobacterium animalis subsp. lactis Probio-M8, while the control group continued consuming regular student milk. The intervention lasted for 2 months. Saliva samples were collected at baseline, 2 weeks, and 2 months. The 2bRAD-M simplified metagenomics technique was used to identify the composition and functional pathways of the oral microbiota. The results indicated that the intervention significantly improved the oral hygiene status of children in the intervention group after health assessments. The proportion of children with “good” oral hygiene significantly increased from 20 cases at baseline to 27 cases over 2 weeks (P<0.05) and then remained elevated (22 cases) over 2 months. Moreover, the number of erupted teeth continued to increase over the observation period (P<0.05), while the incidence of tooth decay remained stable. Therefore, the dental caries was reduced after the probiotic intervention. Microbiome diversity analysis revealed that there was significant structural reshaping. The intervention group exhibited significantly lower α-diversity (Shannon and Simpson indices) over 2 months, compared with the control group (P = 0.001). β-diversity analysis (PCoA using Bray-Curtis distance) with PERMANOVA tests indicated that the intervention and time factors were attributed to the microbial community structure (P=0.001). Notably, there was a more pronounced effect on functional structure, rather than on taxonomic composition, with higher PERMANOVA F-values for the functional profiles. In terms of oral microbiota composition, the intervention group exhibited a significant increase in the relative abundance of Veillonella species, including Veillonella nakazawae and Veillonella rogosae, after 2 weeks and 2 months of intervention. This enrichment remained significantly higher than that of the control group at both time points (P < 0.05). Conversely, the relative abundance of certain Streptococcus species, such as Streptococcus infantis and Streptococcus oralis S, was lower (P < 0.001) than that in the control group over 2 weeks. Streptococcus species, including Streptococcus parasanguinis and Streptococcus symci, showed a slight rebound in relative abundance within the intervention group after 2 months, which remained lower than the levels in the control group at the same time point. Functional analysis of the oral microbiota revealed that the probiotic intervention also modulated multiple metabolic pathways in a time-dependent manner. The intervention group exhibited enrichment in the pathways with iron acquisition (K02014, iron complex outer membrane receptor protein), oxidative stress response (K03711, fur family transcriptional regulator), and substance transport (K03561, biopolymer transport protein ExbB) after 2 weeks post-intervention. Concurrently, the intervention group shared the lower relative abundance of genes encoding amino acid transport systems (K02028-K02030) and ABC transporter-related functions (K01990, K01992), compared with the control group. While K02517 (Kdo2-lipid IVA lauroyltransferase/acyltransferase) remained elevated in the intervention group by the 2-month endpoint. The amino acid and ABC transporters shared the attenuated differences or returned toward control levels. Differentially expressed pathways reflected the dynamic functional adaptation and stabilization of the microbial community. Correlation analysis further revealed that the abundance of Veillonella species, such as Veillonella sp900549845, Veillonella nakazawae, and Veillonella parvula A, was significantly positively correlated with oral hygiene status (r>0), while some Streptococcus species and specific metabolic pathways, such as K07024 (sucrose-6-phosphatase) and K06147 (ATP-binding cassette, subfamily B), were positively correlated with the risk of tooth decay (r>0). In conclusion, the probiotic-enhanced children's formula milk significantly improved preschool children's oral health, particularly in terms of oral hygiene and tooth development, and potentially delayed tooth decay. The intervention also demonstrated a modulating effect on the composition and functionality of the oral microbiota. The microbial diversity can further support the potential of probiotics in oral health. Future studies should explore the long-term probiotic intervention on tooth decay prevention and the stability of the oral microbiota.