Abstract: Objective To investigate the changes in rat cardiac function and myocardium ultrastructure in response to ouabain treatment. Methods Twenty-four male SD rats were randomized into two equal groups to receive daily intraperitoneal injection of ouabain or saline for 4 consecutive weeks, and their systolic blood pressure (SBP) was recorded weekly. After 4 weeks of injection, echocardiography was performed and the hemodynamic parameters were measured by invasive cardiac catheterization, and the changes in myocardium ultrastructure observed using transmission electron microscopy. Results After 4 weeks of ouabain injection, no significant changes in the mean SBP occurred in comparison with the saline group, but echocardiographic examination showed significant increases in the left ventricular end-diastolic and end-systolic diameters, septum thickness, posterior wall thickness, left ventricular mass and isovolumetric relaxation time but significantly lowered E/A ratio, ejection fraction and fractional shortening after ouabain treatment (P<0.05). Invasive monitoring revealed significant attenuation of the left ventricular developed pressure, rate of pressure development (+dp/dt) and rate of pressure decay (-dp/dt), and increment of the left ventricular end diastolic pressure. Myofibrillar fragmentation, swelling of the cardiac myocytes, absence of the Z line, increases of the mitochondria and collagen fibers were found in ouabain group by transmission electron microscopy. Conclusion Ouabain can induce left ventricular enlargement, cardiac wall thickening, myocardial ultrastructural alterations, systolic and diastolic dysfunction in rats before blood pressure elevation is detected, indicating that ouabain can directly cause cardiac damage in rats.