Objective To investigate the expression pattern of Tbx3 in the mouse embryonic second heart field (SHF) to provide morphological basis for the study of the regulation mechanism in SHF. Methods Serial sections of 40 mouse embryonic hearts aged from embryonic day（ED）9 to ED15 were stained by immunohistochemistry or double-label immunofluorescence with antibodies against islet-1 (ISL-1), myosin heavy chain (MHC), myosin light chain 2 a (MLC2a), proliferating cell nuclear antigen (PCNA) and T-box transcription factor 3 (Tbx3). Results Immunohistochemical positive staining for Tbx3 and SHF marker ISL-1 was distributed in pharyngeal ventral endoderm of ED9 mouse. During ED10 to ED12, immunohistochemical positive staining for ISL-1 was presented in pharyngeal ventral mesenchymal and staining intensity was increased gradually. Some ISL-1 positive mesenchymal cells were co-expressed with Tbx3. ISL-1 was also shown in the aortic sac wall, pericardial dorsal wall and the distal pole of the outflow tract, where Tbx3 was negative. However, the dorsal mesenchymal protrusion (DMP) contained ISL-1 or Tbx3 positive cells. During ED13 to ED15, DMP was myocardialized and sporadic Tbx3 positive cells were seen in DMP. Conclusion Tbx3 expression is mainly concentrated in pharyngeal ventral mesenchyme, which suggests that Tbx3 may contribute to the survive and proliferation of SHF progenitor cells. However, its function is different in posterior second heart field（pSHF）and in SHF of arterial pole of embryonic heart.