Cardiac failure due to sepsis is a common cause of death in patients with sepsis, but the specific mechanism leading to cardiac dysfunction is not clear. Therefore, we established a rat model of lipopolysaccharide (LPS)-induced sepsis-mediated cardiac dysfunction to investigate changes in the expression of miR-499 in the plasma and cardiac tissue and the potential mechanism. The results showed that after LPS stimulation, the expression of miR-499 in the plasma and myocardium was downregulated, the expression of α-myosin heavy chain (MHC) in the myocardium was significantly downregulated, and the expression of β-MHC in the myocardium was significantly upregulated. β-MHC/α-MHC dysregulation was reversed after miR-499 was overexpressed with the intravenous administration of a miR-499 agomir. An increased left ventricular ejection fraction (LVEF) , increased cardiac dysfunction and β-MHC/α-MHC dysregulation were observed after the injection of a miR-499 antagomir. These results suggest that miR-499 can improve sepsis-induced cardiac dysfunction in rats by regulating the expression levels of α-MHC and β-MHC, and targeting miR-499 could be an effective approach for treating sepsis-induced cardiac dysfunction.