Background <p>To investigate bilirubin-induced injury in rat myocardial cells at varying concentrations.</p> Methods <p>The study utilized the rat cardiomyocyte cell line H9C2 and primary rat cardiomyocytes. Bilirubin-rich and control sera were prepared, and cells were cultured for 48&#xa0;h with or without vitamin C. Cell viability was assessed using the CCK-8 assay, while superoxide dismutase (SOD), glutathione peroxidase (GPx), Na⁺/K⁺-ATPase, creatine kinase-MB (CK-MB), and cardiac troponin I (cTn-I) levels were measured using their respective assay kits.</p> Results <p>Bilirubin treatment markedly reduced the viability of H9C2 cells and primary rat cardiomyocytes compared to the control group. Additionally, it elevated the levels of cardiac injury markers, including cTn-I and CK-MB in the culture supernatant. Conversely, bilirubin exposure led to a decline in the release of GPx, Na⁺/K⁺-ATPase, and SOD in the medium. Vitamin C supplementation demonstrated partial attenuation of bilirubin-induced effects: including enhanced viability of primary rat cardiomyocytes, partially restored GPx, Na⁺/K⁺-ATPase, and SOD levels, and reduced concentrations of CK-MB and cTn-I in bilirubin-treated cells.</p> Conclusions <p>Hyperbilirubinemia induces concentration-dependent cardiotoxicity in rat models, while vitamin C supplementation partially mitigates bilirubin-induced myocardial damage.</p> Trial registration <p>Not applicable.</p>