Abstract Body: Ferroptosis, an iron-dependent cell death marked by lipid peroxidation, is implicated in various cardiovascular diseases. Duchenne muscular dystrophy-associated cardiomyopathy (DMD-CM) is a leading cause of morbidity and mortality in DMD patients. We have shown that reducing sarcolipin (SLN) ameliorates dystrophic pathology in DMD-CM. However, the role of ferroptosis in DMD-CM remains unexplored. Here, we used dystrophin and utrophin double-knockout (mdx:utrn^–/–) mice with a SLN heterozygous-knockout (sln^+/–) background to study the effect of SLN reduction on ferroptosis susceptibility in DMD-CM. Live/dead viability assays revealed a significantly higher cell death rate in cardiomyocytes from mdx:utrn^–/– (53.7±7.2%, n=24) compared to wild-type (WT) mice (11.7±1.1%, n=17, p<0.01). The selective ferroptosis inhibitor ferrostatin-1 (10 µM) prevented the cell death in mdx:utrn^–/– (7.4±1.5%, n=9, p<0.01), suggesting the involvement of ferroptosis. Notably, ferroptosis was significantly reduced in cardiomyocytes from mdx:utrn^–/–:sln^+/– mice (11.3±2.4%, n=23, p<0.01). Additionally, mdx:utrn^–/– cardiomyocytes showed even higher ferroptosis rates than WT cardiomyocytes when treated with ferroptosis inducers such as iron and the GPX4 inhibitor RSL3. These rates were also reduced in mdx:utrn^–/–:sln^+/– cardiomyocytes. We performed RNA isolation from the ventricles of 3-month-old wild type (WT) and mdx:utr^-/- mice (n=3) and performed RNA-Seq analysis and found alterations of ferroptosis-related pathways. Western blot analysis of ferroptosis-related biomarkers confirmed the upregulation of transferrin receptor and arachidonate 5-lipoxygenase, along with the downregulation of heme oxygenase-1 and ferroptosis suppressor protein 1 in mdx:utrn^–/– hearts, while glutathione peroxidase 4 levels remained unchanged. We also observed an increased level of iron deposition and lipid peroxidation in mdx:utrn^–/– hearts compared to WT. However, the alterations in the aforementioned ferroptosis-related biomarker in mdx:utrn^–/– were not reversed in mdx:utrn^–/–:sln^+/– hearts, suggesting the potential involvement of other pathways presumably Ca²⁺ signaling. Our results suggest that mdx:utrn^–/– exacerbates ferroptosis in cardiomyocytes, while the reducing SLN mitigates this vulnerability. This highlights a potential therapeutic avenue involving SLN reduction to prevent ferroptosis-related complications in muscular dystrophy.