Abstract Body: Doxorubicin (DOX) is a potent chemotherapy drug that has been extensively used in cancer treatment. However, its major side effect, DOX-cardiomyopathy, results primarily from its impacts on mitochondrial energetics and oxidative stress in cardiomyocytes. Recent research demonstrates that kynurenic acid (KynA), a tryptophan metabolite, is cardioprotective against ischemia/reperfusion injury via G protein-coupled receptor 35 (GPR35)-induced ATP synthase inhibition by ATP synthase inhibitory subunit 1 (IF1) to prevent mitochondrial ATP depletion. We hypothesize that KynA is cardioprotective against DOX-cardiotoxicity via GPR35/IF1 signaling (see Figure). Wild type (WT) and IF1 KO (IF1^-/-) (male,C57BL6J) received KynA (3mg/kg) or vehicle for one week, followed by weekly DOX (5mg/kg) or vehicle treatments for four weeks. The echocardiographic assessment confirmed DOX-induced cardiac dysfunction in all mice. However, KynA pretreatment significantly improved cardiac contraction with less declined ejection fraction (EF%) and LDH elevation in all DOX-treated mice. IF1^-/- mice exhibited less reduced EF% and elevated LDH than WT mice, but KynA pretreatment showed no additional benefits. KynA pretreatment markedly attenuated DOX-induced histological (e.g.,cardiac dystrophy,fibrosis) and ultrastructural (e.g., sarcomere degeneration, mitochondrial abnormalities) changes in the heart of DOX-treated mice. IF1^-/- mice exhibited less impaired cardiac pathologies than KynA pretreated WT mice. Western blot analysis revealed that DOX treatment increased cardiac IF1 expression in the vehicle but not KynA-treated mice, suggesting KynA directly or indirectly suppresses cardiac IF1 expression. In cultured neonatal cardiomyocytes (NCM) from WT, KynA significantly reduced DOX-induced cell death and mitochondrial and general reactive oxygen species (ROS) production. Cellular energetic analyses using Seahorse Bioanalyzer showed improved maximal and spare respiration in DOX-treated in IF1^-/- relative to WT NCM. In summary, our study demonstrated that KynA and IF1 ^-/- enhanced mitochondrial function and preserved cardiac structure/function against DOX-induced cardiotoxicity. However, KynA treatment did not provide additive protection in IF1 ^-/- hearts. KynA’s beneficial effects on mitochondria are at least partly by inhibiting DOX-induced cardiac IF1 overexpression. In conclusion, our findings support that KynA may serve as a novel therapy to mitigate doxorubicin-cardiomyopathy.