Objective To investigate the development of the cardiac conduction system of embryonic mouse heart. Methods Serial transverse sections of forty mouse embryonic hearts from embryonic day (ED) 9 to ED 16 were stained immunohistochemically or immunofluorescently with antibodies against hyperpolarization-activated cyclic nucleotide-gated cation 4 (HCN4), myosin heavy chain (MHC), connexin 43(CX43) and podoplanin. Results At ED9, strong positive expression of HCN4 was concentrated in the MHC negative sinus venosus, then gradually shifted to the sinoatrial node during the development of the embryonic heart. From ED11 onward, CX43 negative expression showed site-specific patterns. CX43 negative sinoatrial node was seen extending along the dorsal right atrial wall and left and right venous valves towards the dorsal wall of atrioventricular canal. At ED13, with fusion of base of the CX43 negative septum primum, left and right venous valves continued with the developing atrioventricular node derived from the dorsal wall of the atrioventricular canal. Developing atrioventricular bundle on the top of the interventricular septum was found connecting with atrioventricular node. From ED9 to ED10, strong positive expression of podoplanin was detected both in MHC positive myocardium and in myocardial precursors of splanchnic mesoderm of dorsal pericardial wall and mesenchyme surrounding sinus venosus. Between ED11 and ED13, podoplanin positive mesenchymal cells spread along outer surface of the heart and formed a podoplanin positive mesothelial epicardium. Conclusion At early stage of heart development, the leading pacemaker is located in sinus venous that exhibits function as a pacemaker earlier than contraction. CX43-negative myocardium observed at ED11 represents the establishment of the prototype of the developing cardiac conduction system. Podoplanin is involved in promoting myocardial precursor cells differentiating into cardiocytes.