<p>Myocardial ischemia/reperfusion injury (MI/RI) is a complex state involving numerous inflammatory mediators, which significantly worsens the outcomes of patients with cardiovascular diseases. However, no standardized treatment plan for MI/RI prevails at present. Cucurbitacin B (Cb-B) is a natural bioactive substance extracted from the stem of melon flowers, and is associated with Wnt-β-Catenin, NF-κB, and PI3K/AKT signaling pathways to initiate apoptosis and inflammatory responses in various cancers. Nevertheless, its potential preventive role in MI/RI remains unclear. Here, the potential mechanism adopted by Cb-B for alleviating MI/RI was explored by network pharmacology and in vivo experiments. Firstly, network pharmacology methods, including multiple data retrieval, network construction analysis, gene ontology enrichment analysis, pathway analysis, and molecular docking, were employed to identify the target genes, signaling pathways, and potential mechanisms of Cb-B in the possible treatment of MI/RI. Simultaneously, results were validated through in vivo experiments. The results showed that the comprehensive network pharmacology identified cellular pyroptosis and apoptosis-related genes as <i>nlrp3</i>, <i>caspase1</i>, <i>bcl-2,</i> and <i>caspase3</i>. Functional enrichment analysis showed the association of these target genes with inflammatory response, positive regulation of cytokine production, and response to lipopolysaccharides in biological processes. In addition, the MI/RI rat model showed that Cb-B could alleviate myocardial injury by reducing the release of cellular pyroptosis and apoptosis-related genes. These results suggested that the mechanism of cardioprotective effects of Cb-B may be related to the regulation of PTEN/PI3K/AKT signaling pathways, which lays a foundation for further research on MI/RI treatment.</p>