We report a neonatal case of the use of alteplase for the lysis of a large aortic arch thrombus formed during extracorporeal membrane oxygenation support. Alteplase (0.1–0.15 mg/kg/hour) was infused for thrombolysis, and meanwhile, unfractionated heparin was administrated at 5–10 U/kg/hour for the anticoagulation purpose. Alteplase was successfully administered to this neonate after the repair of congenital diaphragmatic hernia on extracorporeal membrane oxygenation, and the patient survived without apparent catastrophic long-term complications. It is reasonable to consider alteplase therapy during extracorporeal membrane oxygenation support in this setting.