Objective To investigate the relationship between uric acid metabolism and brain injury following cardiopulmonary bypass (CPB) in rats. Methods Healthy male SD rats were randomly assigned to either a Sham group or a CPB group, each comprising 12 rats. The Sham group only underwent vascular puncture and did not perform CPB conversion, while the CPB group was subjected to a CPB procedure with a perfusion duration of 110 min, and the brain tissue was collected post-procedure. Microdialysate was collected 1 h before and after CPB initiation. Apoptosis in the paraventricular nucleus (PVN) was assessed using TUNEL staining, and the expression of Bax mRNA in cerebral cortex and hypothalamus was determined via real-time quantitative PCR. Apoptosis-related protein expression was analyzed by Western blotting. Differentially expressed genes (DEGs) were identified through RNA-sequencing between brain tissues of two groups, and Gene Ontology (GO) analysis was performed to identify enriched pathways among the DEGs. Protein-protein interaction (PPI) networks were constructed using String and Cytoscape softwares to identify key genes. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was employed to analyze differential metabolites in the PVN before and after CPB, with Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis constructed subsequently. Uric acid levels in the hypothalamus was measured using a uric acid assay kit, and the expression of key enzymes of uric acid metabolism [xanthine reductase (XDH), adenosine deaminase (ADA)] and uric acid transporter [organic anion transporter family protein 1 (OAT1), organic anion transporter family protein 3 (OAT3), ATP-binding cassette transporter subfamily G member 2 (ABCG2), glucose transporter 9 (GLUT9)] genes in the hypothalamus was evaluated by real-time quantitative PCR. Results Real-time quantitative PCR revealed a significant upregulation of Bax mRNA in the cerebral cortex and hypothalamus of CPB group compared to Sham group (P<0.05). TUNEL staining indicated a significantly higher apoptosis rate of cells in PVN region in CPB group than that in Sham group (19.0%±5.0% vs. 7.6%±0.8%, P=0.01). Western blotting showed a significantly increased Bcl-2/Bax ratio in the hypothalamus of CPB group compared to Sham group (P<0.05). A total of 2829 DEGs were identified between Sham group and CPB group, with 1374 upregulated genes and 1455 downregulated genes. Uric acid metabolism-related pathways were predominantly enriched in purine nucleoside metabolism and biosynthesis, purine nucleoside monophosphate metabolism, purine nucleoside triphosphate metabolism, purine ribonucleotide metabolism and biosynthesis, purine ribonucleoside monophosphate metabolism and biosynthesis, purine ribonucleoside triphosphate metabolism and biosynthesis, and reaction to purine compounds. Eighteen differential metabolites were identified in the microdialysate, with 13 upregulated and 5 downregulated metabolites. KEGG enrichment analysis identified 7 significantly enriched metabolic pathways, among which the nicotinate and nicotinamide metabolism pathways were closely related to uric acid metabolism. Both RNA-sequencing and LC-MS/MS analysis suggested alterations in uric acid metabolism in CPB groups. Post-CPB, uric acid concentration in the hypothalamic tissue significantly increased (P<0.01), and the expression of XDH and ADA mRNA in the hypothalamus were significantly increased (P<0.05), while the expression of ABCG2, OAT1, OAT3 and GLUT9 mRNA significantly decreased (P<0.001). Conclusion Uric acid metabolism in brain is altered during CPB, which may be an important mechanism for brain injury following CPB.