Objective To investigate the morphological characteristics of the myocardium and epicardium during the septation and remodeling of the atrioventricular canal in the mouse embryonic heart. Methods Serial sections of twenty-five mouse embryos during embryonic day(ED) 10 to ED15 were stained by immunohistochemistry and immunofluorescence with antibodies against myosin light chain 2a (MLC2a), myosin regulatory light chain 2 (MLC-2), T-box transcription factor 3 (Tbx3), and Lymphoid enhancer-binding factor 1 (Lef1). Results Immunohistochemistry and immunofluorescent staining for MLC2a and Tbx3 were positive in the atrioventricular canal myocardium of ED10 to ED15 mice, but for MLC2 was negative. During ED11 to ED12, the epicardium was observed. At ED12 and ED13, the bilateral atrioventricular canal cushions began to fuse with each other and formed the atrioventricular valve. The epicardium derived mesenchymal cells between the atrioventricular myocardium and epicardium were increased, part of which showed the Lef1 immunohistochemistry positive staining. From ED13, some epicardial cells derived from mesenchymal cells transversed through the myocardium to parietal atrioventricular valve. At ED15, the base of the atrioventricular valve was directly connected with the MLC2a positive atrioventricular myocardium. Conclusion The atrioventricular myocardium develops into the myocardium to support the base of the valves of the adult atrioventricular ring. The epicardium provides mesenchymal cells which contribute to the formation of atrioventricular valves.