Cardiac surgery-associated acute kidney injury (CSA-AKI) occurs in 20-30% of patients after heart surgery with cardiopulmonary bypass (CPB). AKI rates are even higher in VA ECMO treated patients. The pathogenesis of CSA-AKI is incompletely understood, and preventative therapies are lacking. We hypothesize that CSA-AKI occurs due to pump related RBC hemolysis and accumulation of cell-free hemoglobin (CFH) and hemopexin in the kidneys. Methods: A new beating heart model of CPB was developed. Rats were used for CPB experiments; blood pressure (BP) was measured by Millar invasive BP catheters, and Glomerular Filtration Rate (GFR) was measured at 24 hours after CPB using the FITC-Sinistrin (FITC-S) method. Western immunoblots were performed to measure hemopexin (Hpx), glutathione peroxidase 4 (GPX4), and β-actin. Urine KIM-1 was measured by ELISA method. KIM-1 and TATA-box Binding Protein 1 (TBP1) mRNA expression were quantified by qRT-PCR. Plasma levels of cell-free hemoglobin (CFH), Hpx, haptoglobin, and IL-6 were measured by ELISA. Immunofluorescence labeling was used to detect hemoglobin (Hb), Hpx, megalin, and KIM-1 in kidneys. Lipid peroxidation was assessed with 4-hydroxynonenal (4-HNE) staining. Results: Compared with sham controls, GFR was reduced 24 hours after CPB and histological kidney sections had higher scores, indicating CPB-induced AKI. Urine KIM-1 protein level and kidney KIM-1 mRNA were increased after CPB. Immunofluorescence staining also showed tubular KIM-1 staining after CPB. Plasma IL-6 levels were increased after CPB, and plasma Hpx increased 24 hours after CPB. Immunofluorescence staining demonstrated colocalized Hpx and megalin in proximal tubules in kidneys, with hemoglobin staining in kidneys after CPB. Finally, ferroptosis was induced by CPB, as evidenced by decreased GPX4 protein and increased 4-HNE staining at 24 hrs. after CPB. Conclusion: CPB induces AKI with evidence of hemoglobin and Hpx deposition in the proximal tubules and ferroptosis.