Objective: In this study, we aimed to explore the effect and mechanism of NPY/Y1 receptor (YIR) 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 Y1R antagonist BIBO3304 by intraperitoneal injection. Forty male C57BL/6J mice were randomly divided into 4 groups: control group (Saline), ISO group (20 mg/kg/day ISO), BIBO3304+ISO group (0.1 mg/kg/day BIBO3304+20 mg/kg/day ISO), and BIBO3304 group (0.1 mg/kg/day BIBO3304). All the drug was administered continuously for 14 days. H9C2 cells were cultured and directly treated with the specific Y1R agonist [Leu31, Pro34]-NPY in vitro. Real-time PCR was used to investigate the expression of NPY, ANP, β-MHC mRNA in the heart of mice and/or in H9C2 cells. Western blot analysis was used to detect the protein expression of NPY, active β-catenin, p-GSK3β and total-GSK3β in the heart and/or H9C2 cells. HE and Masson staining were used to observe the changes of myocardial fiber structure and the degree of myocardial fibrosis. The viability of H9C2 cardiomyocytes was detected by CCK8. Nuclear β-catenin accumulation was detected by immunofluorescence staining. ICG001, a specific β-catenin inhibitor was used to treat H9C2, and then the cardiomyocyte hypertrophy and cell viability induced by [Leu31, Pro34]-NPY were further examined. Results: Compared with the Saline group, cardiac NPY mRNA and protein expressions increased significantly in ISO group (p<0.05), in which it also exhibited myocardial fiber arrangement disorder, cardiomyocyte necrosis, high degree of myocardial fibrosis and increased gene expressions of cardiac hypertrophy. Compared with the ISO group, the myocardial damage and fibrosis were effectively alleviated in BIBO3304+ISO group, and the expressions of cardiac hypertrophy gene decreased significantly (p<0.01). With [Leu31, Pro34]-NPY direct treatment, the level of ANP and β-MHC mRNA expression increased in H9C2, while the cardiomyocytes viability decreased (p<0.01). It showed that the protein expression levels of active β-catenin and p-GSK3β/t-GSK3β increased in the heart of mice from ISO group (p<0.05) and [Leu31, Pro34]-NPY induced H9C2 cells (p<0.05). The administration of BIBO3304 reversed the protein changes (p<0.05). [Leu31, Pro34]-NPY facilitated β-catenin accumulation in the nucleus, while BIBO3304 decreased β-catenin expression in the nucleus. Compared with the [Leu31, Pro34]-NPY group, ICG001 could effectively alleviated cardiomyocyte hypertrophy and cell viability reduction (p<0.01). Conclusion: These findings suggested NPY/Y1 receptor mediated cardiac injury and fibrosis through β-catenin pathway.