Cardiovascular diseases (CVDs) are becoming the major cause of deaths worldwide, and their treatment demands novel therapeutic strategies. In this connection, we have identified p21 activated kinase 1 (PAK1) as a novel therapeutic target for the treatment of myocardial infarction (MI), where its expression is decreased when MI is induced with isoproterenol (ISO), which was brought back normal with pretreatment of Vitex negundo leaf ethanolic extract (VNE). These results were also supported by histopathological studies, cardiac markers, antioxidants, and inflammatory cytokines (NF-κB and IL-1β). Further studies with GC–MS analysis of VNE and in silico experiments confirmed 5,7-dihydroxy-6,4′,-dimethoxy flavonone and 3′,5-dihydroxy-6,7,4′,-trimethoxyflavone are responsible for either maintaining or inducing the expression of PAK1 to protect from MI. Our findings for the first time revealed the use of phytoconstituents in the treatment of MI.