Endothelin‐1(ET‐1) is a potent vasoconstrictor involved in the development of cardiovascular diseases and is an important regulator of heart development. However, the role of ET‐1 in cardiac differentiation of mouse embryonic stem cells (mESCs) and the underlying molecular mechanisms remain poorly understood. In the present study, we showed that ET‐1 significantly up‐regulated gene expression of the cardiac specific transcriptional factors Nkx2.5, GATA4, and conduction system specific marker CX40, with no affect on the gene expression of α‐MHC and β‐MHC in cardiac differentiation of mESCs. The percentage of beating embryoid bodies (EB) and the Troponin T (TnT) positive area in total EBs was unchanged following ET‐1 treatment, while the percentage of spindle cells that stained positively with TnT was increased in the presence of ET‐1. Further investigation indicated that the percentage of beating EBs and the TnT positive area were decreased by the extracellular signal‐related kinases (ERK)‐1/2 inhibitor U0126 and the p38 inhibitor SB203580, but not by the Jun amino‐terminal kinases (JNK) inhibitor SP600125. Inhibition of ERK1/2, p38, and JNK pathways also blocked the up‐regulation of Nkx2.5 and GATA4 by ET‐1, however only inhibition of the ERK1/2 pathway had negatively effects on the increase in CX40 expression in response to ET‐1. ET‐1 induced an increase in the percentage of spindle cells was also inhibited by U0126. Our results suggest that ET‐1 plays a significant role in the cardiac differentiation of mESCs, especially in those cells committed to the conduction system, with the ERK1/2 pathway playing a critical role in this process. J. Cell. Biochem. 111: 1619–1628, 2010. © 2010 Wiley‐Liss, Inc.