PURPOSE: Doxorubicin (DOX), an anthracycline antibiotic, has limited clinical use due to its pronounced cardiotoxicity. Irisin, a myokine known for its metabolic regulation, has shown therapeutic effects on cardiovascular disease. This study investigates the potential cardioprotective function of irisin in reducing the cardiac injury induced by DOX. METHODS: In vitro, H9c2 cells were pretreated with irisin (20 nM) for 24 hours before exposure to DOX (1 μM). In vivo, C57BL/6 mice were administered DOX (5 mg/kg/week, i.p.) for 4 weeks, reaching a cumulative dose of 20 mg/kg. Irisin (1 mg/kg/ 3 days, i.p.) was administered to the mice both 7 days prior to and during DOX injection.Cardiac function was evaluated by echocardiography, and cardiac histology was assessed using HE, WGA, and Masson staining. Myocardial injury markers were quantified using ELISA, and apoptosis was analyzed via TUNEL staining. Oxidative stress was determined by measuring antioxidase activity, MDA and GSH levels, and DHE staining, while mitochondrial superoxide production was assessed using MitoSOX Red. Mitochondrial morphology and function evaluated using transmission electron microscopy and Seahorse analysis, respectively Inflammatory cytokines were quantified in serum and cell supernatants. The role of the PERK-eIF2α-ATF4 pathway mediated by irisin was investigated by Western blot. Using adeno-associated virus serotype-9 carrying mouse FNDC5 shRNA (AAV9-shFNDC5) further validated the protective role of irisin in DOX-induced myocardial injury. RESULTS: Irisin reduced DOX-induced cardiac dysfunction and fibrosis. Moreover, irisin mitigated oxidative stress and inflammation through inhibiting the PERK-eIF2α-ATF4 pathway activated by DOX, thus preserving mitochondrial function. While cardiac FNDC5 knockdown exacerbated DOX-induced heart injury and PERK-eIF2α-ATF4 activation, which was partially reversed by irisin. CONCLUSION: Irisin mitigates oxidative stress and inflammation by modulating the PERK-eIF2α-ATF4 pathway, highlighting its potential as a prospective approach for combating DOX-induced cardiotoxicity.