Activated RhoA and ROCK is associated with many cardiovascular diseases (CVD) such as congestive heart failure (CHF), atherosclerosis and hypertension. However, the role of RhoA/ROCK2 signaling pathway in initiating diabetic cardiomyopathy (DCM) has not been fully elucidated. Here, we studied the role of RhoA/ROCK2 signaling pathway in induction of DCM through autophagy suppression in diabetic rat animal models. Broadly, we investigated the potential role and mechanism of diabetes induced myocardial dysfunction in rats. DCM was induced by injections of streptozocin (STZ) in experimental Wistar rats. The experimental rats were randomized to be treated with fasudil and lentivirus carrying the RhoA cDNA. Haemodynamic changes, assessment of cardiac weight index, histopathological examinations, cardiomyocyte autophagy and expression of RhoA and ROCK2 mRNAs were compared between groups. The expression of RhoA and ROCK mRNAs was found significantly increased in cardiac tissues compared with control group. The RhoA overexpression significantly decreased the values of left ventricular ejection fraction (LVEF), ±dp/dt max and repressed autophagy. RhoA/ROCK2 signaling pathway repressed autophagy in diabetic cardiomyopathy indicating that it may serve as a potential therapeutic target for DCM treatment