ETHNOPHARMACOLOGICAL RELEVANCE: The Tongmai Yangxin Pill (TMYX) is derived from the Zhigancao decoction recorded in Treatise on Cold Damage Disorders (Shang Han Lun) by Zhang Zhongjing during the Han dynasty. The prescription of TMYX reflects a therapeutic rationale and efficacy unique to traditional Chinese medicine. TMYX is clinically effective in alleviating myocardial ischemia-reperfusion injury (MI/RI). However, the precise active ingredients and underlying mechanisms remain unclear. AIM OF THE STUDY: The primary objective of this study was to investigate the potential of TMYX in addressing MI/RI by activating the estrogen receptor ERα. We hypothesized that this action upregulates PGC-1α activity, subsequently promoting a balanced regulation of mitochondrial fusion and fission. MATERIALS AND METHODS: UPLC-Q-TOF-MS/MS was used to identify the active components of TMYX. Subsequently, a network pharmacology approach was used to uncover the therapeutic targets and underlying pharmacological mechanisms through which TMYX mitigates MI/RI. Lastly, the anticipated outcomes were confirmed through in vivo and in vitro experimental validations. RESULTS: Using UPLC-Q-TOF-MS/MS, we successfully identified 53 distinct compounds in TMYX. Network pharmacology analysis revealed 20 key TMYX targets associated with MI/RI. Enrichment studies using GO and KEGG analyses revealed that these targets were mainly associated with mitochondrial processes and estrogen signaling pathways. Both in vivo and in vitro studies confirmed that TMYX markedly improved mitochondrial function through the ERα/PGC-1α signaling cascade, leading to a reduction in the size of myocardial infarctions and the incidence of apoptosis. Notably, combining TMYX with siERα abolished the protective effect of TMYX on the mitochondria. CONCLUSION: TMYX therapy can improve cardiac function in MI/RI. This effect is likely mediated by the ERα/PGC-1α signaling pathway. However, given the complex multi-component composition of traditional Chinese medicine formulas, additional studies are necessary to confirm the findings of this research.