Mitochondrial dysfunction is a fundamental challenge in myocardial injury. Ginsenoside Rg1 (Rg1) is a bioactive compound with pharmacological potential for cardiac protection. Optic atrophy 1 (OPA1) acts as a mitochondrial inner membrane protein that contributes to the structural integrity and function of mitochondria. This study investigated the protective role of Rg1 in septic cardiac injury from the perspective of OPA1 stability. Rg1 protected cardiac contractive function against endotoxin injury in mice by maintaining mitochondrial cristae structure. In cardiomyocytes, lipopolysaccharide (LPS) evoked mitochondrial fragmentation and destruction of mitochondrial biogenesis, which were prevented by Rg1, possibly due to the preservation of the integrity of cristae structure. In support, the beneficial effects of Rg1 on cardioprotection and mitochondrial biogenesis were diminished by OPA1 deficiency subjected to the LPS challenge. Mechanistically, LPS stimulation triggered intracellular glutathione destabilization that promoted S-glutathionylation of OPA1 at Cys551, leading to the dissociation of OPA1-Mitofilin. Rg1 interacted with Glutathione S-transferase pi (GSTP1) to inhibit its mediated S-glutathionylation of OPA1, thereby promoting OPA1-Mitofilin interaction and protecting mitochondrial cristae structure. These findings suggest that GSTP1/OPA1 axis may be a beneficial strategy for the treatment of myocardial injury, and expand the clinical application of Rg1.