AIM: Ginsenosides have notable bioactivity in treating cardiovascular diseases, but the mechanisms of their combined use with Peroxiredoxin 6 (PRDX6) in myocardial injury remain unclear. This study explores the synergistic effects of Ginsenoside Rb1 (Gs-Rb1) and PRDX6, aiming to provide a theoretical foundation for their therapeutic potential. METHODS: We established a rat model of isoproterenol (ISO)-induced myocardial injury and observed that combination therapy was more effective than single-drug treatments, as shown by ECG monitoring and Masson staining. We performed RNA sequencing (RNA-Seq) on the combination therapy group and the ISO group. The results indicated that, compared to the ISO group, the combination therapy alleviated myocardial injury by reducing inflammation, oxidative stress, and apoptosis. Further analyses, including cell morphology, apoptosis rates, HE staining, ROS fluorescence intensity, and inflammation-related proteins, confirmed that the combination therapy successfully inhibited apoptosis, managed oxidative stress, and lessened inflammation. RESULTS: Combined treatment with Gs-Rb1 and PRDX6 significantly inhibited cardiac tissue fibrosis in rats, leading to a marked decrease in serum CK and LDH levels. RNA-seq analysis revealed upregulated genes related to lipid metabolism and small molecule biosynthesis, while downregulated genes were associated with oxidative stress, inflammation, and apoptosis. Validation experiments confirmed the combined treatment's significant inhibition of apoptosis, ROS activity, and inflammation. These results support the effectiveness of the two-drug combination in suppressing key biological processes in cardiac tissue, suggesting potential mechanisms for combating cardiac fibrosis. CONCLUSION: This study clarifies how Gs-Rb1 and PRDX6 work together to protect against myocardial damage, demonstrating that their combined therapy reduces inflammation, apoptosis, and oxidative stress. This highlights a new avenue for developing ginseng-based treatments.