Heart failure (HF) is a cardiovascular disease with an extremely high mortality rate. However, Morinda officinalis How (MO) has not been studied for cardiovascular purposes at this time, the aim of this study was to find new mechanism for the MO of treatment of HF through a bioinformatics and experimental validation. The present study also aimed to establish a link between the basic and clinical applications of this medicinal herb. MO compounds and targets were obtained by traditional Chinese medicine systems pharmacology (TCMSP) and Pubchem. Subsequently, HF targets were acquired from DisGeNET and the interactions of all the targets and other human proteins were obtained via String so as to establish a component-target interaction network by Cytoscape 3.7.2. All the targets of clusters were inserted into Database for Annotation, Visualization and Integrated Discovery (DAVID) to perform GO (gene ontology) enrichment analysis. Molecular docking was adopted to predict the targets of MO relevant to the treatment of HF and to further explore the associated pharmacological mechanisms. Subsequently, a series of in vitro experiments, including histopathological staining, immunohistochemical and immunofluorescence analyses were conducted for further verification. Moreover, western blot analysis and in vivo experiments were performed. The results indicated that MO alleviated apoptosis, regulated cholesterol metabolism and transport function, and reduced inflammation, which resulted in the successful treatment of HF. Beta-sitosterol, Asperuloside tetraacetate and americanin A were the key bioactive components of MO. ALB, AKT1, INS, STAT3, IL-6, TNF, CCND1, CTNNB1, CAT, and TP53 were the core potential targets, which were significantly associated with multiple pathways, namely the FoxO signaling pathway, the AMPK signaling pathway, and the HIF-1 signaling pathway. In vivo experiments validated that MO may protect against heart failure or treat this disease by increasing the levels of autophagy via the FoxO3 signaling pathway in rats. The present study suggested that a combination of network pharmacology prediction with experimental validation may offer a useful tool to characterize the molecular mechanism of action of the traditional Chinese medicine (TCM) MO in the treatment of HF.