OBJECTIVES: Iron overload (IO) cardiomyopathy is a major cause of mortality in patients with iron overload disorders. This study investigates the role of cystathionine γ-lyase (CSE), a key enzyme in the transsulfuration pathway for cysteine and hydrogen sulfide (H2S) production, in iron-induced oxidative cardiac injury. METHODS: We investigated the effects of CSE on oxidative stress, metabolic dysregulation, and cardiac remodeling using in vivo mouse models of chronic iron overload and in vitro ferric citrate (FAC)-treated cardiomyocytes. RESULTS: Genetic deletion of Cth (encoding CSE) in mice exacerbated iron overload-induced cardiac hypertrophy, systolic dysfunction, and interstitial fibrosis. These effects correlated with reduced expression of the cysteine transporter SLC7A11, impaired glutathione (GSH) synthesis, and suppression of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/glutathione peroxidase 4 (GPX4) antioxidant signaling pathway. Conversely, CSE overexpression in cardiomyocytes restored SLC7A11 stability, and suppressed reactive oxygen species (ROS) production through the Nrf2/HO-1/GPX4 pathway, thereby attenuating iron-mediated oxidative damage. Mechanistically, CSE deficiency disrupted both cysteine uptake via SLC7A11and cysteine production via transsulfuration pathway, inducing a self-reinforcing cycle of oxidative stress. CONCLUSION: These findings demonstrate that the CSE-transsulfuration pathway serves as a crucial regulator of cysteine metabolism and antioxidant signaling in cardiomyocytes, presenting a potential therapeutic target in iron-induced cardiomyopathy.