Objective To study the protective effects and mechanism of sacubitril (Sac)/valsartan (Val) on cardiomyocytes of rabbits with heart failure induced by doxorubicin (DOX). Methods Thirty New Zealand rabbits were selected to establish DOX-induced heart failure rabbit model. Twenty-five rabbits with successful modeling were randomly assigned to model group (DOX group, n＝9), DOX＋Val group (n＝8), and DOX＋Sac/Val group (n＝8); and another 8 New Zealand rabbits were selected as blank group. The DOX＋Val group was gavaged with 4.65 mg/kg Val suspension each time, the DOX＋Sac/Val group was gavaged with 9.3 mg/kg Sac/Val suspension each time, and the blank group and DOX group were gavaged with equal volume of distilled water each time. Each group was gavaged twice a day for 8 weeks. After 8 weeks of administration, echocardiography was used to measure left ventricular end-diastolic diameter (LVDD), left ventricular end-systolic diameter (LVSD), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS). The heart mass index (HMI) and left ventricular mass index (LVMI) were calculated. The pathological morphology and myocardial fibrosis of myocardial tissue were observed by hematoxylin-eosin (H-E) and Masson staining. The ultrastructure of cardiomyocytes was observed by transmission electron microscope. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was used to observe cardiomyocytes apoptosis and apoptosis rate was calculated. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), high-sensitivity cardiac troponin I (Hs-cTNI), angiotensinⅡ (AngⅡ), aldosterone (ALD), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), cyclic guanosine monophosphate (cGMP), and protein kinase G (PKG) in serum. Quantitative polymerase chain reaction (qPCR) was used to detect the expression of natriuretic peptide receptor A (NPR-A), cGMP-specific phosphodiesterase 5A (PDE5A［cGMP］), PKG, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), and cysteine aspartate protease 3 (caspase 3) mRNA in myocardial tissue. Western blotting was used to detect the expression of phosphorylated cAMP response element-binding protein (p-CREB), phosphorylated Bcl-2 related death promoting factor (p-Bad), Bcl-2, Bax, and caspase 3 proteins in myocardial tissue. Results Compared with the blank group, the LVDD and LVSD in the DOX group were increased (both P＜0.01), the LVEF and LVFS were decreased (both P＜0.01) and the HMI and LVMI were increased (both P＜0.01); the apoptosis and apoptosis rate of cardiomyocytes were increased (P＜0.01); the levels of NT-proBNP, Hs-cTNI, AngⅡ, ALD, ANP, BNP, cGMP and PKG and the expression of NPR-A, PDE5A (cGMP), PKG, p-CREB, Bax and caspase 3 were all increased (all P＜0.01), while the expression of Bcl-2 was decreased (P＜0.01), and the expression of p-Bad had no significant difference (P＞0.05). Compared with the DOX group, the LVDD and LVSD of the DOX＋Sac/Val group and DOX＋Val group were decreased (all P＜0.01), the LVEF and LVFS were increased (all P＜0.01) and the HMI and the LVMI were decreased (all P＜0.01); the apoptosis and apoptosis rate of cardiomyocytes were decreased (all P＜0.01); the levels of NT-proBNP, Hs-cTNI, Ang Ⅱ, ALD, ANP, BNP, cGMP and PKG and the expression of NPR-A, PDE5A (cGMP), PKG, Bax and caspase 3 were all decreased (all P＜0.01), while the expression of Bcl-2 was increased (P＜0.01); and the expression of p-CREB and p-Bad was increased in the DOX＋Sac/Val group (both P＜0.01), but there was no significant difference in the DOX＋Val group (both P＞0.05). Compared with the DOX＋Val group, the DOX＋Sac/Val group showed a decrease in all indicators except for LVEF, LVFS, NPR-A, ANP, BNP, cGMP, PDE5A (cGMP), PKG, p-CREB, p-Bad, and Bcl-2, which were all elevated (all P＜0.05). Myocardial pathology and transmission electron microscopy showed that Sac/Val effectively protected cardiomyocytes, reduced cardiomyocytes apoptosis and myocardial fibrosis, and these effects were significantly better than those of Val. Conclusion Sac/Val can effectively reduce cardiomyocytes apoptosis, improve cardiac function and reduce myocardial fibrosis in rabbits with heart failure, and these effects are superior to Val. Its mechanism may be related to activating the NPR-A/cGMP/PKG signaling pathway and inhibiting renin-angiotensin-aldosterone system.