Background: Moderate hypothermic circulatory arrest (MHCA) is a safe and effective method of cardiopulmonary bypass (CPB). However, most present rat models involve a deep hypothermic circulatory arrest, which cannot exactly reflect the clinical situation. The aim of this study was to establish a novel and safe rat model of MHCA with hyperkalemia-induced cardioplegia to study the pathophysiology of potential complications. Methods: Ten adult male Sprague–Dawley rats (age, 16–18 weeks; weight, 450–550 g) were used. The entire CPB circuit consisted of a reservoir, peristaltic pump, membrane oxygenator, heat exchanger, and hemoconcentrator, all of which were connected via silicon tubing. The prime solution was approximately 19 mL. The right jugular vein, right femoral artery, and left femoral artery were cannulated. Blood was drained from the right atrium through the right jugular vein and perfused to the rats via the left femoral artery. CPB was commenced at a full flow rate. The rats were cooled to a rectal temperature of 25°C, and cardioplegia was induced by systemic hyperkalemia. After that, MHCA was carried out for 30 min. At the same time, system self-ultrafiltration was carried out to decrease the concentration of potassium by a hemoconcentrator. The circulatory arrest was followed by reperfusion and over 30 min of rewarming. CPB carefully was terminated. Blood in the circuit slowly was centrifuged for autotransfusion. Blood gas and hemodynamic parameters were recorded at each time point before CPB, before MHCA, at 10 min after the initiation of rewarming, and after CPB. Results: All CPB and MHCA procedures successfully were achieved. One rat died of respiratory failure. Cardioplegia with systemic hyperkalemia was induced by 1 mL of 10% potassium chloride injected into the reservoir, and the concentration of potassium was maintained at 17 ± 3 mmol/L. Cardiac function and blood pressure were stable after the operation. Conclusions: A novel and safe rat model of MHCA with hyperkalemia-induced cardioplegia successfully was established.