Background <p>Myocardial infarction (MI) triggers oxidative stress, mitochondrial dysfunction, and cardiomyocyte apoptosis. At present, it remains urgently needed to develop novel therapies specifically suppressing cardiomyocyte apoptosis via improving mitochondrial dysfunction following MI. Jiedu Huoxue Decoction (JDHXD) may have the effect of ameliorating myocardial injury after MI.</p> Purpose <p>This study examined the protection exerted by JDHXD against myocardial injury post-MI and investigated the underlying mechanisms of action.</p> Methods <p>UHPLC/Orbitrap-MS, network pharmacology and transcriptome analysis were used to study the effective components and potential targets of JDHXD for treating MI. In vivo: The MI mice received JDHXD (12.74/25.48&#xa0;g/kg/day) or captopril treatment for 28&#xa0;days. Later, cardiac function (tested by echocardiography and histopathology), apoptosis, oxidative stress, mitochondrial ultrastructure, mitochondrial fission/fusion and PTEN/AKT/GSK3β pathway protein levels were evaluated. In vitro: TBHP-induced cardiomyocytes (H9C2 cells and NCMs) were exposed to JDHXD treatment (50–200&#xa0;μg/mL), with or without PTEN inhibitor Bpv (2&#xa0;μM) or PTEN overexpression (through adenoviral transduction). Afterwards, cell apoptosis, oxidative stress, mitochondrial membrane potential, and relative proteins were assayed. Experiments such as molecular docking and surface plasmon resonance imaging (SPRi) were conducted to verify the effective components of JDHXD in preventing myocardial mitochondrial injury after MI.</p> Results <p>The results of network pharmacology and transcriptomics suggest that JDHXD may ameliorate myocardial injury after MI through modulating PTEN for activating the PI3K/AKT/GSK3β signaling pathway. In vivo: JDHXD dose-dependently improved left ventricular function, improved the oxidative stress-induced imbalance of mitochondrial fission/fusion, and inhibited cardiomyocyte apoptosis post-MI in association with suppressing the PTEN/AKT/GSK3β pathway. In vitro: JDHXD suppressed the TBHP-induced cardiomyocyte apoptosis, attenuated oxidative stress, preserved mitochondrial potential, and restored mitochondrial dynamics. PTEN inhibitor did not augment JDHXD’s effects, whereas PTEN overexpression partially abolished JDHXD’s protection against myocardial injury induced by oxidative stress. UHPLC/Orbitrap-MS, molecular docking, SPRi and experiments in vitro confirmed that puerarin is one of the main components of JDHXD in regulating the PTEN/AKT/GSK3β pathway to improve mitochondrial function after MI and inhibit cardiomyocyte apoptosis.</p> Conclusions <p>JDHXD against oxidative stress-induced cardiomyocyte apoptosis post-MI through ameliorating mitochondrial dysfunction, which is partially mediated by suppressing the PTEN/AKT/GSK3β pathway to inhibit excessive mitochondrial fission and promote mitochondrial fusion.</p> Graphical Abstract <p></p>