Objective:To investigate the protective effects and underlying mechanisms of carbon monoxide-releasing molecule-3 (CORM-3) against hypoxia-reoxygenation-induced injury in Caco-2 cells.Methods:A hypoxia-reoxygenation injury model was established in Caco-2 cell monolayers by culturing cells under glucose-free, serum-free hypoxic conditions (1% O 2) for 8 hours, followed by reoxygenation with glucose and serum restoration for 4 hours. Experimental groups included: blank control (Control), hypoxia-reoxygenation model (HR), and HR with 300 μmol/L (HR_C1), 400 μmol/L (HR_C2), or 500 μmol/L CORM-3 (HR_C3), plus HR with 500 μmol/L inactive iCORM-3 (HR_iC). CORM-3/iCORM-3 was administered during the hypoxic phase. Assessments included: cell viability (CCK-8 assay), apoptosis/necrosis rates (flow cytometry), monolayer permeability (sodium fluorescein), tight junction protein distribution (ZO-1, Occludin, Claudin2 immunofluorescence), protein expression (Western blot), and transcriptomic analysis of differentially expressed genes. Results:Compared to controls, HR group showed significantly reduced cell viability ( P<0.05), increased apoptosis/necrosis rates, elevated sodium fluorescein permeability (both P<0.05), disrupted tight junction structure, decreased Occludin and increased Claudin2 expression (both P<0.05). CORM-3 treatment significantly improved viability ( P<0.05), reduced apoptosis/necrosis rates and permeability (both P<0.05, concentration-dependent), mitigated tight junction damage, showed a non-significant trend toward increased Occludin, and significantly decreased Claudin2 expression ( P<0.05). Transcriptomics identified 25 differentially expressed genes, with KEGG analysis revealing 14 significant pathways (including MAPK signaling, inflammatory bowel disease, and cellular senescence). GO analysis highlighted immune-inflammatory responses and cell membrane barrier components, with TGFB3 as the primary immune-related gene. Conclusions:CORM-3 effectively reduces apoptosis/necrosis, preserves tight junctions, and mitigates hypoxia-reoxygenation injury in Caco-2 cells, potentially through MAPK signaling regulation and immune-inflammatory response modulation.