BACKGROUND: Mitochondrial dynamics imbalance is an essential pathological mechanism in heart failure (HF). The Chinese herbal formula Xin-Ji-Er-Kang (XJEK) has demonstrated good therapeutic effects in various cardiovascular disease models. However, whether XJEK treats HF by regulating mitochondrial dynamics homeostasis and its specific molecular mechanisms remain elusive. PURPOSE: To investigate the effect of XJEK on restoring the disrupted mitochondrial dynamics homeostasis in HF and elucidate the potential regulatory mechanism. STUDY-DESIGN/METHODS: A mouse model of myocardial ischemia-reperfusion (MIR)-induced HF was established to assess the cardioprotection of XJEK. Subsequently, network pharmacology was employed to predict the mechanism by which XJEK treated HF. Moreover, gene silencing was employed to explore the potential mechanisms behind the cardioprotective effects of XJEK in AC16 cells subjected to hypoxia/reoxygenation (H/R). RESULTS: XJEK treatment significantly attenuated myocardial fibrosis and ameliorated ventricular remodeling in post-MIR-induced HF mice. Network pharmacology analysis identified the estrogen receptor α (ERα) as a key regulator of XJEK-mediated cardioprotection. XJEK disordered mitochondrial dynamics in the hearts of MIR-induced HF mice. In addition, XJEK restored mitochondrial fusion-fission imbalance and facilitated ERα nuclear translocation to up-regulate sirtuin 3 (SIRT3) expression in the hearts of MIR-induced HF mice and H/R-induced AC16 cells. Notably, ERα depletion in cardiomyocytes completely abrogated the cardioprotective effects of XJEK. CONCLUSION: XJEK safeguards the hearts in mice with MIR-induced HF by facilitating ERα nuclear translocation to up-regulate SIRT3 expression to rescue the mitochondrial fusion-fission imbalance. This study establishes a new theoretical basis for treating HF with XJEK.