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Myocarditis is a clinical condition characterized by inflammation of the myocardium. It commonly occurs in various heart disorders and, in extreme circumstances, can be fatal. However, the effective and safe dietary strategies for alleviating myocardial inflammatory injury are currently lacking. Here we investigated the potential of daidzein (DD), a natural isoflavone found in leguminous plants, in alleviating myocardial inflammatory injury. The lipopolysaccharides (LPS)-treated C57BL/6J mice and H9c2 cardiomyocytes were employed to investigate the protective effects and mechanisms of DD against myocardial inflammation. In animal studies, DD supplementation at 50 and 100 mg/kg/day significantly alleviated the myocardial injury induced by LPS. In cell experiments, 2.5 μM DD effectively decreased LPS-induced oxidative stress, cell death, and inflammatory responses. In addition, DD mitigated the LPS-induced calcium imbalance in cardiomyocytes. Mechanistically, DD inhibited the LPS-induced myocardial inflammatory injury by targeting estrogen receptor beta (ERβ), subsequently leading to a reduction in ER stress. Furthermore, the molecular docking and experimental validation revealed that the binding pocket of DD in ERβ was highly conserved, and that the H475 and R376 residues were essential for binding DD. In summary, our results suggest that dietary DD supplementation is a promising strategy for alleviating myocardial inflammatory injury.