Aim： To investigate the effects of the cardiotonic steroid, ouabain, on cardiac differentiation of murine embyronic stem cells （mESCs）. Methods： Cardiac differentiation of murine ESCs was enhanced by standard hanging drop method in the presence of ouabain （20 pmol/L） for 7 d. The dissociated ES derived cardiomyocytes were examined by flow cytometry, RT-PCR and confocal calcium imaging. Results： Compared with control, mESCs treated with ouabain （20 pmol/L） yielded a significantly higher percentage of cardiomyocytes, and significantly increased expression of a panel of cardiac markers including Nkx 2.5, α-MHC, and β-MHC. The α1 and 2- isoforms Na^＋/K^＋-ATPase, on which ouabain acted, were also increased in mESCs during differentiation. Among the three MAPKs involved in the cardiac hypertrophy pathway, ouabain enhanced ERK1/2 activation. Blockage of the Erkl/2 pathway by U0126 （10 pmol/L） inhibited cardiac differentiation while ouabain （20 pmol/L） rescued the effect. Interestingly, the expression of calcium handling proteins, including ryanodine receptor （RyR2） and sacroplasmic recticulum Ca^2＋ ATPase （SERCA2a） was also upregulated in ouabain-treated mESCs. ESC-derived cardiomyocyes （CM） treated with ouabain appeared to have more mature calcium handling. As demonstrated by confocal Ca^2＋ imaging, cardiomyocytes isolated from ouabain-treated mESCs exhibited higher maximum upstroke velocity （P〈0.01） and maximum decay velocity （P〈0.05）, as well as a higher amplitude of caffeine induced Ca^2＋ transient （P〈0.05）, suggesting more mature sarcoplasmic reticulum （SR）. Conclusion： Ouabain induces cardiac differentiation and maturation of mESC-derived cardiomyocytes via activation of Erk1/2 and more mature SR for calcium handling.