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Myocardial infarction (MI) is a life-threatening ischemic disease and is one of the leading causes of morbidity and mortality worldwide. Serotonin (5-HT) release during myocardial ischemia plays an important role in the progression of myocardial cellular injury. This study was conducted to investigate the possible cardioprotective effect of flibanserin (FLP) against isoproterenol (ISO)-induced MI in rats. Rats were randomly divided into five groups and were treated orally (p.o.) with FLP (15, 30, and 45 mg/kg) for 28 days. ISO was administered subcutaneously (S.C.) (85 mg/kg) on the 27th and 28th days to induce MI. ISO-induced myocardial infarcted rats exhibited a significant increase in cardiac markers, oxidative stress markers, cardiac and serum 5-HT levels, and total cardiac calcium (Ca2+) concentration. ISO-induced myocardial infarcted rats also revealed a remarkable alteration of electrocardiogram (ECG) pattern and significantly upregulated expression of the 5-Hydroxytryptamine 2A (5-HT2A) receptors gene. Moreover, ISO-induced myocardial infarcted rats showed significant histopathological findings of MI and hypertrophic signs. However, pretreatment with FLP significantly attenuated the ISO-induced MI in a dose-dependent manner, as the effect of FLP (45 mg/kg) was more pronounced than that of the other two doses, FLP (15 and 30 mg/kg). The present study provides evidence for the cardioprotective efficacy of FLP against ISO-induced MI in rats.