Objective  To investigate the effects of insulin-induced acute hypoglycemia of rats on the incidence of cardiac arrhythmia and expression of myocardial connexin43 (Cx43). Methods  The model of diabetic rat (DM) was established by intraperitoneal injection with streptozotocin (STZ). After the model was successfully established, rats were randomly divided into the diabetic hyperglycemia group (DM group, n=9) and diabetic hypoglycemia group (DMHY group, n=9), while the same batch of normal rats were randomly divided into the normal blood glucose group (Sham group, n=9) and hypoglycemia group of normal rats (NHY group, n=9). Rats of the DMHY group and NHY group were transjugularly infused with insulin to induce hypoglycemia. The thorax was opened and arrhythmias was induced by programmed electrophysiological stimulation (PES). Serum epinephrine and norepinephrine concentrations were measured and expressions of myocardial Cx43 of each group were detected by the Western blotting. Results  Compared with the Sham group, the blood glucose level of the DM group significantly increased, body weight greatly decreased, and the induced rate of ventricular arrhythmias significantly increased (P<0.05). The induced rate of ventricular arrhythmias of the NHY group greatly increased and ventricular fibrillation threshold (VFT) significantly decreased (P<0.05). Compared with the DM group, the induced rate of ventricular arrhythmias of the DMHY group significantly increased and VFT greatly decreased (P<0.05). The descending order of the expression of myocardial Cx43 of each group was that of the Sham group, DM group, NHY group, and DMHY group. The differences between hypoglycemia groups and corresponding non-hypoglycemia groups were statistically significant (P<0.05). Compared with corresponding non-hypoglycemia groups (Sham group and DM group), epinephrine and norepinephrine levels of hypoglycemia groups (NHY group and DMHY group) significantly increased (P<0.05). Conclusion  Insulin-induced acute hypoglycemia contributes to the incidence of ventricular arrhythmias and the mechanism may be relevant to the decrease of VFT and myocardial Cx43 expression.