Background <p>Leonurus japonicus Houtt. (LJH) has multiple pharmacological effects.</p> Objective <p>To investigate the potential mechanism of LJH in the treatment of myocardial ischemia-reperfusion injury (MIRI) using network pharmacology, molecular docking technology, and in vitro experiments.</p> Methods <p>Herbs for ischemic heart disease were identified with the help of herb-disease databases. The TCMSP database was used to find the potential targets of LJH. Disease targets of MIRI were identified with the help of Disgenet, Genecard, Alliance of Genome Resources databases. The common targets were obtained with the help of VENN diagram, and the common targets were analyzed by GO function and KEEG pathway enrichment to predict the potential mechanism of action of LJH in treating MIRI. With the help of STRING database and Cytoscape software, we constructed a visual protein-protein interaction (PPI) network model to screen the core targets and then docked the core targets with the corresponding ligand molecules. AC16 cells were used to simulate MIRI by glucose-oxygen deprivation, and apoptosis was detected by Annexin V-FITC/PI double staining; protein expression was detected by Western blot.</p> Results <p>LJH was one of the herbal remedies for the treatment of ischemic heart disease. LJH had 247 potential targets of action and 26 targets in common with MIRI. These 26 targets were enriched in the TNF signaling pathway and NF-kappa B signaling pathway, and the core targets screened by the PPI results included TNF, VCAM1, and MMP9. Molecular docking results showed that the compounds in LJH docked well with the core target proteins. In vitro experiments showed that LJH could inhibit the elevation of TNF, VCAM1, and MMP9 after MIRI, reduce apoptosis, and inhibit inflammation.</p> Conclusion <p>The mechanism of LJH in the treatment of MIRI was mainly related to the activation of TNF signaling pathway and NF-kappa B signaling pathway, and the regulation of TNF, VCAM1, and MMP9 protein expression.</p>