Embryonic stem cells (ESCs) can differentiate into functional cardiomyocytes and thus represent a promising cell source for cardiac regenerative therapy. Nevertheless, the therapeutic application of ESC-derived cardiomyocytes is limited by the low efficacy of the current protocol for cardiac differentiation and their immature phenotypes. Although thyroid hormone is essential for normal cardiac development and function, its role in the cardiac differentiation of ESCs, as well as the maturation of ESC-derived cardiomyocytes, remains unclear. In this study, we examined the cardiac differentiation of murine ESCs in the presence of T(3) for 7 d using flow cytometry, RT-PCR, cellular electrophysiology study, and confocal calcium imaging. Compared with control conditions, T(3) supplementation increased the number of ESC-derived cardiomyocytes and was accompanied by up-regulation of a panel of cardiac markers, including Nkx2.5, myosin light chain-2V, as well as alpha- and beta-myosin heavy chain. More importantly, electrophysiological study revealed that ESC-derived cardiomyocytes exhibited more adult-like phenotypes after T(3) supplementation based on action potential characteristics. They also exhibited more adult-like calcium homeostasis properties. These phenotypic changes were associated with up-regulation of sarco(endo)plasmic reticulum calcium ATPase-2a and ryanodine receptor-2 expression. In addition, the classical (genomic) pathway was shown to be involved in T(3)-induced cardiac differentiation of ESCs. Our results show that T(3) supplementation promotes cardiac differentiation of ESCs and enhances maturation of electrophysiological, as well as calcium homeostasis, properties of ESC-derived cardiomyocytes.