BACKGROUND: Perform proteomic and metabolomic analysis on bilateral renal pelvis urine of patients with unilateral calcium oxalate kidney stones to identify the specific urinary microenvironment associated with stone formation. METHODS: Using cystoscopy-guided insertion of ureteral catheters, bilateral renal pelvis urine samples are collected. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is employed to identify differential proteins and metabolites in the urine microenvironment. Differentially expressed proteins and differential metabolites are further analyzed for their biological functions and potential metabolic pathways through Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, Reactome pathway analysis and Biomolecular Interaction Network Database protein-protein interaction (PPI) network analysis. RESULTS: In the urine from the stone-affected side, 36 differential proteins were significantly upregulated, 4 differential proteins were downregulated, and 10 differential metabolites were significantly upregulated. Functional and pathway analyses indicate that the differentially expressed proteins are primarily involved in inflammatory pathways and complement and coagulation cascades, while the differential metabolites are mainly associated with oxidative stress. CONCLUSION: The proteomic and metabolomic profiles of the urinary microenvironment in stone-affected kidneys provide a more precise reflection of the pathophysiological mechanisms involved in stone formation and development.