Aim To study the effect of TNNI3K gene on differentiation of mouse embryonic stem cells into cardiomyocytes. Methods Firstly, we identified the omnipotency of mESC by the morphological characteristics and cellular immunofluorescence, alkaline phosphatase test (ALP) and HE staining. In addition, Embryonic body, cultured by hanging drop method, differentiated spontaneously into beating cardiomyocytes, which were identified by cellular immunofluorescence and transmission electron microscopy (TEM). Furthermore, hTNNI3K gene and siRNA, carried by Lentivirus, infected mESC more than 10 days, respectively, and spontaneously differentiated into mature cardiomyocytes, the differences of myocardial marker proteins expression levels were ananlyzed by flow cytometry (FCM), cellular immunofluorescence, Western blot. Results The membrane proteins of mESC, SSEA-1 and Oct-4, were presented green fluorescence, and ALP test presented blue-purple as well as HE staining with karyoplasmic ratio ＞＞1. What’s more, it was cTnⅠ, cTnT, MLC2 and α-actinin that were clearly visible by cellular immunofluorescence, as well as the unique structure of the muscle fibers by TEM. Furthermore, the cTnT⁺ positive cells rate of TNNI3K-overexpression group was remarkably higher than that in control group, likely the expression of cTnⅠ, MLC2, GATA4, et al. Interestingly, the contraction protein MHC6 expressed earlier than other groups. The rate of cTnT⁺ positive cells in the siRNA group was significantly lower than that of the control group, the same as the expression of other cardiac specific proteins. Conclusions TNNI3K gene might enhance the synthesis of cardiomyocytes and promote the differentiation of mESC into cardiomyocytes.