Abstract: This study investigated the intervention effect and possible mechanism of ophiopogonin D (OPD) in protecting cardiomyocytes against ophiopogonin D' (OPD')-induced injury, and provided relevant experimental data for the clinical use of Ophiopogon japonicas. Cell counting kit-8 (CCK-8) assay was used to evaluate the effect of OPD and OPD' on H9c2 cell viability. The content of reaction oxygen species (ROS) in cells were detected by flow cytometry. The contents of Fe²⁺ in cells were detected by FerroOrange's fluorescence imaging. The content of glutathione (GSH) and glutathione peroxidase (GSH-Px) were detected by kits. The expression of transferrin receptor 1 (TFR1), cyclooxygenase 2 (COX2), NADPH oxidase 1 (NOX1), long-chain acyl-CoA synthetase 4 (ACSL4), cationic amino acid transporter 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and ferritin heavy chain 1 (FTH1) was detected by Western blot. Results showed that OPD' (1 μmol·L^-1) significantly induced the expression of ferroptosis-related proteins, the contents of Fe²⁺, ROS, and GSH-Px were increased, and the content of GSH were decreased. In addition, different concentrations of OPD (0.5, 1, and 2 μmol·L^-1) could partially reverse the myocardial cell injury caused by OPD', and the best effect was obtained when the dose range was 1-2 μmol·L^-1. The experimental results show that OPD can interfere with the ferroptosis caused by OPD', and then have a protective effect on H9c2 cells.