The presence of 5-HT7r’s in both human and rat cardiovascular and immune tissues and their contribution to inflammatory conditions prompted us to hypothesize that these receptors contribute in acute myocardial infarction (MI) with underlying chronic endothelial dysfunction. We investigated the role of 5-HT7 receptors on heart tissue that damaged by isoproterenol (ISO)-induced MI in rats with high-fat diet (HFD). In vitro and in vivo effects of 5-HT7r agonist (LP44) and antagonist (SB269970) have been investigated on the H9C2 cell line and rats, respectively. For in vivo analyses, rats were fed with HFD for 8 weeks and after this period ISO-induced MI model has been applied to rat. To investigate the role of 5-HT7r’s, two different doses of LP44 and SB269970 were evaluated and compared with standard hypolipidemic agent, atorvastatin. In vitro studies showed that LP44 has protective and proliferative effects on rat cardiomyocytes. Also in in vivo studies stimulating 5-HT7r’s by LP44 improved blood lipid profile (decreased total cholesterol, low-density lipoprotein-C, and triglyceride, increased high-density lipoprotein), decreased cardiac damage markers (creatine kinase and troponin-I), and corrected inflammatory status (tumor necrosis factor-α, interleukin-6). Our results showed significant improvement in LP44 administered rats in terms of histopathologic analyses. In damaged tissues, 5-HT7 mRNA expression increased and agonist administration decreased this elevation significantly. We determined for the first time that 5-HT7r’s are overexpressed in ISO-induced MI of rats with underlying HFD-induced endothelial dysfunction. Restoration of this overexpression by LP44, a 5-HT7r agonist, ameliorated heart tissue in physiopathologic, enzymatic, and molecular level, showing the cardiac role of these receptors and suggesting them as future potential therapeutic targets.