Neuregulin-1 (NRG-1) is a multifunctional regulator that acts through receptor tyrosine kinases of the epidermal growth factor (EGF/ErbB) receptor family in diverse tissue. ErbB receptors are expressed in developing embryoid bodies (EBs), and the importance of the NRG-1/ErbB signaling axis in heart development has been investigated, but the underlying molecular mechanism is poorly studied. NRG-1 treatment at 100 ng/ml significantly increased the number of beating EBs of differentiated murine embryonic stem cells (ESCs). Furthermore, NRG-1 up-regulated the expression of the cardiac-restricted transcription factors Nkx2.5 and GATA-4 and factors involved in differentiated cardiac cells (alpha-MHC, beta-MHC and alpha-actinin); NRG-1-induced increase of Nkx2.5 transcription was inhibited by treatment with the PI3 K inhibitor or ErbB receptor inhibitor. Western blot analysis confirmed that the expression of phospho-Akt in the beating foci was increased in the presence of NRG-1. Our results suggest that NRG-1 promotes cardiomyocyte differentiation of ESCs and the ErbB/PI3 K/Akt signaling pathway is one of the underlying molecular mechanisms.