Sodium/proton exchanger 1 (NHE1) plays an important role in the cardiomyocyte development. To study the effect of NHE1 activity in stem cells differentiation into cardiomyocytes, we treated P19 stem cells with dimethyl sulfoxide (DMSO) to initiate cardiomyocyte differentiation. In separate experiments, P19 cells were incubated with NHE1 specific inhibitor EMD87580 during the DMSO induction. The formed embryoid bodies (EBs) were detected with cell morphology detection, immunohistochemisty staining and RT-PCR analysis of expression of cardio-specific gene markers. Results showed that P19 cells were able to differentiate into cardiomyocytes and form the beating cell clusters. However, when cells treated with NHE1 inhibitor EMD87580, they could still form the EBs and proliferate when cell clusters adhered on the culture plate, but cells were unable to differentiate. This observation indicates that inhibition of NHE1 activity affected P19 stem cells differentiating into cardiomyocytes.