Abstract Body: Doxorubicin (DOX) is a chemotherapeutic agent used to treat cancer, but its clinical use is restricted due to cardiotoxicity. Senescence is characterized by resistance to apoptosis and a senescence-associated secretory phenotype (SASP). Elimination of senescent cells can improve cardiac dysfunction, suggesting that senescent cardiomyocytes (CMs) contribute to impaired heart function. Although DOX induces senescence in CMs, little is known about the mechanisms mediating DOX-induced senescence and cardiomyopathy. Wnt5a, a non-canonical Wnt ligand, is an important player in cardiac diseases and is upregulated in induced pluripotent stem cell (iPSC)-derived CMs from patients with heart failure induced by DOX chemotherapy. However, the role of Wnt5a in mediating DOX-induced cardiomyopathy remains elusive. Wnt5a protein expression was increased (4.4-fold, p<0.001) in mouse hearts with DOX-induced cardiomyopathy. Upregulation of Wnt5a following DOX treatment was also observed in neonatal rat CMs (NRCMs), and the degree of upregulation was more prominent in CMs (2.1-fold) than in non-CMs (1.1-fold). In NRCMs, Wnt5a induced senescent phenotypes, including increases in the p16 protein level, the number of gH2AX foci per cell and SA-b-gal activity. Wnt5a knockdown reduced DOX-induced increases in the p16 protein level in NRCMs, suggesting that Wnt5a mediates DOX-induced CM senescence. To assess the role of endogenous CM Wnt5a in mediating DOX-induced cardiomyopathy, we generated Myh6-cre Wnt5a^fl/fl (Wnt5a^cKO) mice, in which Wnt5a is knocked out in a CM-specific manner. DOX-induced upregulation of Wnt5a in the heart was completely abrogated in Wnt5a^cKO mice, suggesting that DOX-induced Wnt5a upregulation in the heart occurs primarily in CMs. Notably, there was little difference in ejection fraction (EF) between DOX-treated and PBS-treated Wnt5a^cKO mice (56.64 ± 6.17% and 57.60 ± 5.23%), while DOX-treated Wnt5a^fl/fl mice showed a significant decrease in EF (34.63 ± 3.65%, p<0.0001). There was little difference in cardiac function between PBS-treated Wnt5a^cKO and Wnt5a^fl/fl mice, indicating that the absence of Wnt5a in CMs did not alter baseline cardiac function. These results suggest that Wnt5a deletion in CMs impedes the progression of DOX-induced cardiac dysfunction, positioning Wnt5a as a novel therapeutic target for the prevention of DOX-induced cardiomyopathy.