Objective To explore the relationship between the development of pulmonary endoderm-associated second heart field (PSHF) and the morphogenesis of the distal outflow tract in mouse embryonic heart. Methods The islet-1 (ISL-1) expression in 80 mouse embryonic hearts from embryonic days (ED) 10 to ED 14 was detected by Western blotting. Both the immunohistochemistry and immunofluorescence staining method were used to observe ISL-1, α-smooth muscle actin (α-SMA）and myosin heavy chain (MHC) distribution in serial sections of 36 mouse embryos from ED 11 to ED 13. Results The peak period of ISL-1 protein expression in mouse embryonic heart was from ED 11 to ED 12. At ED 11, the ISL-1 positive cells from branchial arch or dorsal wall of pericardium, belonging to PSHF, extended into the distal outflow tract, which lost MHC expression and showed α-SMA positive. The ISL-1 positive cells from PSHF formed thecone-shaped structure centered by pulmonary endoderm, which protruded into aortic sac and produced the ISL-1 positive protrusion in aortic sac. At ED 11.5, though aortic sac was still not separated, the extension of PSHF to the cranial and caudal myocardial wall of aortic sac was detected as two ISL-1 positive symmetrical boluses in outflow tract wall. Instead of MHC, the protrusion of PSHF became α-SMA expression at ED 12. Before the fusion of PSHF protrusion and outflow tract cushions, a small channel called the aortic-pulmonary foramen was observed. By later ED 12, PSHF protrusion completed fusion with outflow tract cushions generated the α-SMA positive and transient aortic-pulmonary septum, which divided aortic sac into the intrapericardial aorta and pulmonary trunks which were MHC negative. At ED 13, the aortic-pulmonary septum gradually disappeared, and the intraper-ardial aorta and pulmonary trunks separated finally, which were MHC negative and in which α-SMA positive smooth muscle layers were observed. The extension of a few ISL-1 positive cells from PSHF toward the intrapericardial aorta and pulmonary trunks walls was continuing. Conclusion From ED 11 to ED 13 in normal mouse embryos, PSHF divides aortic sac into the intrapericardial aorta and pulmonary trunks, and is responsible for the lateral and facing walls of intrapericardial trunks.