Objective：To explore the effect and mechanism of neuropeptide Y（NPY）/Y1 receptor signaling in myocardial injury. Methods：The C57BL/6J mice model of cardiac injury was established by subcutaneous injection with isoproterenol（ISO），which was subsequently treated with the specific NPY/Y1 receptor antagonist BIBO3304 by intraperitoneal injection. C57BL/6J mice were randomly divided into 4 groups：control group（Saline），ISO group［20 mg/（kg · d）ISO］，BIBO3304 + ISO group［0.1 mg/（kg · d） BIBO3304+20 mg/（kg·d）ISO］，and BIBO3304 group［0.1 mg/（kg·d）BIBO3304］. All the drug was administered continuously for 14 days. The expression of NPY mRNA and protein in the heart of mice were detected by qPCR and Western blot，respectively. The change of myocardial fiber structure and myocardial fibrosis were observed by HE and Masson staining. Levels of cardiac hypertrophy related gene，atrial natriuretic peptide（ANP）and β-myosin heavy chain（β-MHC）mRNA in the heart of mice were also estimated by qPCR.［Leu31，Pro34］-NPY，a specific Y1 receptor agonist，was used to directly treat H9C2 cells in vitro. The mRNA levels of ANP and β-MHC in the cardiomyocytes were detected by qPCR，and the cell viability of H9C2 was measured by CCK-8. The expressions of active β-catenin，phospho-glycogen synthesis kinase 3β（p-GSK3β）and total glycogen synthesis kinase 3β（t-GSK3β）in the heart and H9C2 cells were analyzed by Western blot. Nuclear β- catenin accumulation was estimated by immunofluorescence staining. ICG001，a specific β-catenin inhibitor，was used to treat H9C2，and the cardiomyocyte hypertrophy and cell viability induced by［Leu31，Pro34］- NPY were further examined. Results：Compared with the control group，cardiac NPY mRNA and protein expressions were increased significantly in ISO group（P < 0.05），in which it exhibited myocardial fiber arrangement disorder，high degree of myocardial fibrosis and increased expression of cardiac hypertrophy related gene. Compared with the ISO group，the myocardial damage and fibrosis were effectively alleviated in BIBO3304+ISO group，and the expression of cardiac hypertrophy related gene were decreased（P < 0.01）. Compared with the control group，［Leu31，Pro34］-NPY increased ANP and β-MHC mRNA levels， and decreased cell viability in H9C2 cardiomyocytes（P < 0.01）. Compared with the control group，the protein expressions of active β- catenin and p-GSK3β/t-GSK3β were increased in the heart of mice from ISO group（P < 0.05）. Compared with the ISO group，the expressions of active β-catenin and p-GSK3β were decreased in BIBO3304+ISO group（P < 0.05）. Compared with the control group， ［Leu31，Pro34］-NPY increased expressions of active β-catenin and p-GSK3β，as well as facilitated nuclear β-catenin accumulation in the cardiomyocytes（P < 0.05）. Compared with［Leu31，Pro34］-NPY group，BIBO3304 decreased β-catenin expression in the nucleus， and ICG001 effectively alleviated cardiomyocyte hypertrophy and cell viability reduction（P < 0.01）. Conclusion：NPY/Y1 receptor mediates cardiomyocyte injury and fibrosis through β-catenin pathway.