Abstract: Objective To investigate the feasibility of inducing ES cells into cardiomyocytes by 5-azacytidine (5-aza) in vitro. Method P19 cells were seeded into 24 well plates or culture dishes containing a thin layer of agar. After exposed to 5-aza of 5μmol/L for 7 days, the cells were cultured without 5-aza. The beating of cells was observed under inverted phase contrast microscope. Transmission electron microscopy, immunocyochemistry test and RT-PCR analysis were also used to identify cell differentiation. Result Combination 5-aza treatment and suspension culture can induce P19 cells differentiate toward spontaneously contracting cardiomyocytes. The cells expressed cardiomyocyte specific GATA-4、α-MHCmRNA and α-sarcomeric actin、cTnT protein. The myofiber inside the cells was shown by transmission electron microscopy. Conclusion 5-aza can induce ES cells differentiated into cardiomyocytes in vitro by exposure to 5-aza. The method of suspension culture was suitable to cardiomyocyte differentiation.