Ventricular arrhythmias are commonly observed in patients with acute coronary occlusion and ischemia. The purpose of the present study is to determine ischemic electrophysiological effects and their role in ischemia-induced arrhythmia. Optical mapping of the membrane potential with voltage-sensitive dyes was used in the study. The mapping was performed with di-4-ANEPPS in Langendorff-perfused rabbit hearts. The excitation–contraction decoupler 2,3-butanedione monoxime was used to suppress motion artifacts caused by contraction of the heart. The acute global ischemia was developed by a rapid reduction of the flow rate. The experiments revealed that ischemic tissues were characterized by an obvious reduction in action potential duration and action potential upstroke, slower conduction velocity (CV) and the property of post-repolarization refractoriness. Moreover, the magnitude of CV reduced both in control and ischemia when the pacing cycle length was short. CV reduction was even early in ischemia, resulting in a broader curve during ischemia. Moreover, the dominant frequency of ventricular tachycardia/ventricular fibrillation (VT/VF) in ischemia was less than that in control, implying a decreasing tendency of VT/VF frequency for low excitability. Therefore, combined with our previous simulation study, the dynamic changes of CV and longer refractory period were suggested to play an important role in the ischemia-related arrhythmia. Low excitability in ischemic tissue was the fundamental mechanism in it.