Objective To explore the early development of the outflow tract of the embryonic human heart. Methods Serial sections of twenty-nine human embryonic hearts from Carnegie stage 10 to Carnegie stage 16 were stained immunohistochemically with antibodies against α-SMA (α-smooth muscle actin), α-SCA（α-sarcomeric actin）, MHC (myosin heavy chain) and active caspase3 to investigate the relationship of splanchnic epithelium lining the dorsal wall of the pericardial cavity, the prepharyngeal mesenchyme and the aortic sac with the embryogenesis of the outflow tract myocardium. Results With the caudal translocation of the aortic sac and outflow tract relative to the pharyngeal arches during C10 to C15 and the dorsal expansion of the pericardial cavity on both lateral sides of the outflow tract, the aortic sac originally embedded in the prepharyngeal mesenchyme gradually protruded into the pericardial cavity. The pericardial splanchnic epithelium covering the mesenchymal wall of the aortic sac was found to differentiate progressively into α-SCA and MHC positive cardiomyocytes of the outflow tract. The prepharyngeal mesenchyme migrating to the dorsal and ventral walls of the arterial pole of the outflow tract was seen apoptosed at C15,and the outflow tract cardiomyocytes were detected to proliferate, migrate into and replace the apoptosed outflow tract mesenchymal masses. α-SMA positive cells began to appear in the outflow tract cushions at C12 and gradually aggregated to form two opposite spiral ridges. During C15 and C16, α-SMA positive cells in the posterior wall of the aortic sac proliferated and grew into the aortic sac to form the aortopulmonary septum that divided the aortic sac into the intrapericardial ascending aorta and pulmonary trunk. Conclusion The splanchnic mesodermal epithelium of the pericardial cavity is the secondary heart field of the human embryonic heart, the continuous differentiation of which into cardiomyocytes brings about the increase in the length of the myocardial outflow tract. Cas3 positive staining suggests that not all of the prepharyngeal mesenchymal cells migrating to the arterial pole of the outflow tract can differentiate into cardiomyocytes. α-SMA positive cells in the outf