Hydrogen sulfide (H₂S) has been recently recognized as an important gasotransmitter with cardioprotections, and iron is vital for various cellular activities. This study explored the regulatory role of H₂S on iron metabolism and mitochondrial functions in cultured rat cardiac cells. Rotenone, a mitochondrial complex I inhibitor, was used for establishing an in vitro model of ischemic cell damage. It was first found that rotenone induced oxidative stress and lipid peroxidation and decreased mitochondrial membrane potential and ATP generation, eventually causing cell death. The supplement of H₂S at a physiologically relevant concentration protected from rotenone-induced ferroptotic cell death by reducing oxidative stress and mitochondrial damage, maintaining GPx4 expression and intracellular iron level. Deferiprone, an iron chelator, would also protect from rotenone-induced ferroptosis. Further studies demonstrated that H₂S inhibited ABCB8-mediated iron efflux from mitochondria to cytosol and promoted NFS1-mediated Fe-S cluster biogenesis. It is also found that rotenone stimulated iron-dependent H₂S generation. These results indicate that H₂S would protect cardiac cells from ischemic damage through preserving mitochondrial functions and intracellular Fe-S cluster homeostasis.