Neuregulin-1 (NRG-1) is a vital factor involved in heart development. NRG-1 up-regulated connexin-40 (Cx40) in mice fetal cardiomyocytes has been reported, while the effect of NRG-1 on expression of connexins in embryonic stem cells-derived cardiomyocytes (ESCMs) is limited studied. The process of cardiomyocytes differentiated from embryonic stem cells with or without NRG-1 treatment was observed continuously. Exposure to NRG-1 increased the expression of Cx40 and connexin-45 (Cx45) in ESCMs, while the expression of connexin-43 was unchanged regardless of NRG-1 treatment Western blot analysis also confirmed that the expression of Cx40 and Cx45 in the beating foci was increased in the presence of NRG-1. These results indicate that connexins are differentially regulated by exogenous NRG-1 during cardiomyocytic differentiation of embryonic stem cells.