T-box transcription factor Tbx18 (Tbx18) was expressed in epicardial cells and controls the differentiation of epicardial epithelial cells into myocardial lineage in the developing mouse embryo. Epithelial-to-mesenchymal transition (EMT) plays a crucial role in embryonic development. For the purpose of determine whether Tbx18 regulates downstream EMT-associated signaling molecules, such as the transcriptional factors Snail1, Slug, Smad, Twist and cell adhesion molecule E-cadherint in mouse heart development. In this study, we used the Tbx18-Cre/Rosa26R-EYFP double-heterozygous mice and Tbx18 mutants to investigate the spatiotemporal expressions patterns of the major EMT-associated signaling molecules within Tbx18 lineage cells. We observed that the major EMT-associated transcriptional factors co-localized with Tbx18 lineage cells in the epicedium and subepicardial mesenchyme. The expression patterns of the major EMT-associated signaling molecules within Tbx18 lineage cells were consistent with Tbx18. Most important, we further demonstrate that the four EMT-associate transcriptional factors are reduced and E-cadherin is significantly increased in the Tbx18 mutant hearts compared with the wild type hearts. Our data together indicated that Tbx18 regulated the major EMT-associated major signaling molecules in mouse heart development. Understanding the signaling pathways by which Tbx18 regulates mouse heart development may help to improve regeneration in adult heart disease.