Objective <p>This study aimed to characterize gut microbiota-derived faecal metabolites and evaluate their associations with serum biochemical indices and tumor markers in gastric cancer patients residing in high-altitude regions, using untargeted metabolomics.</p> Methods <p>Stool samples from 30 newly diagnosed gastric cancer patients and 30 healthy controls from Qinghai Province were analyzed using LC–MS-based untargeted metabolomics. Serum biomarkers—including proteins, lipids, and tumor markers—were concurrently measured. Multivariate analysis, fold-change filtering, and correlation analysis were used to identify differential metabolites and their associations with clinical phenotypes. False discovery rate (FDR) correction was applied to reduce false positives.</p> Results <p>A total of 281 faecal metabolites were identified, predominantly lipids (35.4%) and organic acids (29.1%). Significant metabolic alterations were observed in gastric cancer patients, with notable upregulation of glycylproline, glycine, and hydroxyisocaproic acid, and downregulation of cytidine, 5′-methylthioadenosine, and trehalose. Correlation analysis revealed hydroxyisocaproic acid and glycine were positively associated with serum albumin, while 5′-methylthioadenosine was negatively correlated with HDL, LDL, and alpha-fetoprotein. Annotation was supported by MS/MS spectral matching and database scoring. Limitations included a modest sample size, limited control for high-altitude confounders, and lack of targeted validation.</p> Conclusion <p>Gastric cancer patients living at high altitudes exhibit distinct gut microbiota metabolic profiles compared to healthy individuals. Specific faecal metabolites show significant associations with key serum biomarkers, suggesting a microbiota-metabolism-serum axis potentially influenced by environmental and pathological factors. These findings may inform biomarker discovery and future mechanistic studies focused on high-altitude cancer biology</p>