Objective: This study aimed to investigate the regulatory effects of Lacticaseibacillus paracasei LPB27, a strain isolated from infant intestines, on gut microbiota and elucidate its underlying mechanisms, thereby providing a scientific basis for its potential development as a novel probiotic agent. Methods: A total of 48 male BALB/c mice were randomly assigned to four groups: A control group and low-dose, medium-dose, and high-dose intervention groups, following the guidelines outlined in the "Test Methods for Regulating Intestinal Flora" from the ''Functional Evaluation Procedures and Test Methods for Health Foods (2023 Edition)''. The animals were administered LPB27 probiotic powder via gavage at doses of 0, 0.08, 0.17, and 0.50 g/kg body weight, respectively, over a period of 21 days. Body weight changes and alterations in target intestinal bacterial populations in fecal samples were monitored throughout the experiment. The composition and differential microbial species within the gut microbiota were analyzed using 16S rDNA high-throughput sequencing, while PICRUSt2 was employed for predictive functional profiling. Additionally, the concentrations of fecal short-chain fatty acids (SCFAs) were quantified. Results: Following the 21-day intervention, all mice remained in good health with consistent weight gain observed across groups. Notably, significant increases in the levels of Lactobacillus and Bifidobacterium were detected in the medium- and high-dose groups, particularly in the high-dose group. No significant changes were observed in the abundance of pathogenic bacteria, including Enterobacter, Enterococcus, and Clostridium perfringens. These findings confirm the efficacy of LPB27 in modulating intestinal flora in the animal model. Further analysis using 16S rDNA sequencing and SCFA profiling revealed that LPB27 may exert its beneficial effects by modulating key genera such as Akkermansia and Ruminococcus, reshaping the microbial community structure, and enhancing SCFA metabolic pathways. Specifically, butyrate production was significantly elevated in the high-dose group. Conclusion: Lacticaseibacillus paracasei LPB27 demonstrates effective regulation of gut microbiota through a synergistic "microbiota-metabolite" network, involving activation of the L-Methionine Biosynthesis III pathway, enrichment of beneficial genera such as Akkermansia and Ruminococcus, and increased production of beneficial metabolites, particularly butyrate. This study provides a solid scientific foundation for the development of LPB27 as a novel probiotic formulation aimed at regulating intestinal microecology.