Abstract: AIM To investigate the roles of miRNA-1 and miRNA-133 in myocardial differentiation from mouse pluripotent stem cells.METHODSExpression profiles of miRNA-1 and miRNA-133 during myocardial differentiation of miPSCs were analyzed by RT-PCR. miPSCs were infected by adenovirus overexpression vector of miRNA-1 and miRNA-133 individually, which were divided into four groups: control group, miRNA-1 group,miRNA-133 group and miRNA-1+miRNA-133 group. Expressions of miRNA and cardiac-specific genes and protein were detected by RT-PCR, q-PCR and immunofluorescence. RESULTSExpressions of miRNA-1 and miRNA-133 were upregulated during the last stage of cardiac differentiation. The forced expression of miRNA-1 or miR-133 alone using lentiviral vectors in miPCRs did not promote myocardial differentiation of miPSCs. However, overexpression of miRNA-1 with miRNA-133 increased the number of beating cells and expression of cardiac genes and promoted the maturity of the myocardial cells from miPSCs. CONCLUSIONmiRNA-1 and miRNA-133 may play a synergistic role in promoting the differentiation of mouse pluripotent stem cells into cardiomyocytes in vitro, thus increasing the efficiency of cardiac differentiation.