The probiotic yeast Saccharomyces boulardii exhibits remarkable tolerance to gastrointestinal stress conditions, yet the specific metabolic adaptations enabling this resilience remain incompletely characterized. In the present study, we employed comprehensive LC-MS-based metabolomic profiling to investigate S. boulardii's dynamic metabolic responses to simulated gastric fluid (SGF), intestinal fluid (SIF), and sequential SGF-SIF exposure. Our systematic analysis revealed that organic acids, lipids, and organoheterocyclic compounds constituted the predominant metabolic classes affected by gastrointestinal stress, with carboxylic acids and their derivatives representing the most significantly altered subclass. Notably, intestinal fluid exposure induced the most profound metabolic perturbations, resulting in quantitative changes to 2,029 distinct metabolites, including 1,008 upregulated and 1,021 downregulated species. A detailed pathway analysis (KEGG) demonstrated significant activation of purine/pyrimidine metabolism and amino sugar/nucleotide sugar biosynthesis pathways, which are fundamentally important for maintaining cellular energy status and cell wall integrity during stress exposure. Conversely, tyrosine metabolism and taurine-related pathways showed marked downregulation, suggesting potential metabolic trade-offs during stress adaptation. Through an advanced correlation network analysis, we identified Parisvanioside A and specific glyceryl phosphatides as key metabolites positively associated with stress tolerance, while valorphin and D-glucosamine exhibited substantial depletion under stress conditions. These findings provide compelling evidence for S. boulardii's metabolic flexibility during gastrointestinal transit and reveal specific molecular targets for probiotic optimization. The identified metabolic signatures suggest promising strategies for enhancing probiotic viability, including targeted metabolite stabilization or nutritional supplementation approaches during gastrointestinal passage.