Background/Objectives: Obesity is closely associated with gut microbiota dysbiosis, intestinal barrier dysfunction, and impaired glucose and lipid metabolism. However, single probiotic or prebiotic interventions often yield only limited metabolic improvements. This study aimed to evaluate the effects of a synbiotic formulation comprising Lacto-N-tetraose (LNT) and Bifidobacterium animalis subsp. lactis MN-Gup (MN-Gup) in a high-fat diet (HFD)-induced obese mouse model. Methods: In this study, an HFD-induced obese mouse model was used to investigate whether the synbiotic formulation of LNT and MN-Gup could ameliorate obesity-related metabolic dysregulation, intestinal barrier dysfunction, and gut microbiota imbalance. Mice were treated with LNT alone, MN-Gup alone, or the synbiotic at different doses. Serum biochemical parameters, glucose tolerance, lipid profiles, liver histopathology, intestinal barrier markers, gut microbiota composition, short-chain fatty acid (SCFA) levels were analyzed. Results: High-dose synbiotic intervention significantly outperformed single-component treatments in reducing weight gain, improving glucose tolerance and lipid profiles, and attenuating hepatic lipid accumulation and injury in mice. These metabolic changes were accompanied by improved markers of intestinal barrier integrity and modulation of gut microbiota composition, characterized by the enrichment of beneficial genera (e.g., Akkermansia, Leuconostoc, and Alistipes) alongside a reduction in obesity-associated taxa (including Desulfovibrionaceae_unclassified, Colidextribacter, Helicobacter, Erysipelatoclostridium, Peptococcaceae_unclassified, and Firmicutes_unclassified). Spearman correlation analysis revealed associative links between microbial alterations and host metabolic markers. Conclusions: Collectively, these findings suggest that the synbiotic formulation comprising high-dose LNT and MN-Gup offers potential benefits for managing high-fat diet-induced metabolic dysregulation in mice.