Myocardial injury is the most prevalent and serious complication of sepsis. The potential of puerarin (Pue) to treat sepsis-induced myocardial injury (SIMI) has been recently reported. Nevertheless, the specific anti-SIMI mechanisms of Pue remain largely unclear. Integrating network pharmacology, bioinformatics analysis, and experimental validation, we aimed to clarify the anti-SIMI mechanisms of Pue, thereby furnishing novel therapeutic targets. Pue-associated targets were collected from HIT, GeneCards, SwissTargetPrediction, SuperPred, and CTD databases. SIMI-associated targets were acquired from GeneCards and DisGeNET. Differentially expressed genes (DEGs) were identified from GEO database. Potential anti-SIMI targets of Pue were determined using VennDiagram. ClusterProfiler was employed for GO and KEGG analyses. STRING database and Cytoscape were used for protein-protein interaction (PPI) network construction, and cytoHubba was used for hub target screening. PyMOL and AutoDock were utilized for molecular docking. An <i>in vitro</i> SIMI model was built to further verify the therapeutic mechanisms of Pue. Seventy-three Pue-SIMI-DEG intersecting target genes were obtained. GO and KEGG analyses revealed that the targets were principally concentrated in cellular response to chemical stress, response to oxidative stress (OS), and insulin and neurotrophin signaling pathways. Through PPI analysis and molecular docking, AKT1, CASP3, TP53, and MAPK3 were identified as the pivotal targets. <i>In vivo</i> experiments indicated that Pue promoted cell proliferation, downregulated AKT1, CASP3, TP53, and MAPK3, and inhibited inflammation, myocardial injury, OS, and apoptosis in the cell model. Pue might inhibit inflammation, myocardial injury, OS, and apoptosis to treat SIMI by reducing AKT1, CASP3, TP53, and MAPK3.