Objective To analyze the features of P19 cells differentiating into cardiomyocyte after induced by 5-azacytidine(5-aza) in vitro. Methods The P19 cells were cultivated in the medium containing different concentrations(1,5 and 10μmol/L) of 5-aza in dishes laid with a thin layer of agar to form embryonic bodies(EBs). On the 7th day, EBs were transferred to coverslips in 24 well plates, allowed to adhere and be cultured in the medium without 5-aza. The beating of cells was observed. α-sarcomeric actin and cardiac Troponin T(cTnT) immunofluorescence stain and GATA-4, α-MHC gene RT-PCR analysis were used to identify cell differentiation. Results After the 5-aza induced EBs were cohesively cultured, spontaneously and rhythmically beating cells were found within the EBs outgrowths, which were α-sarcomeric actinpositive and cTnTpositive, and expressed GATA4, α-MHC gene simultaneously. The treatment with 10μmol/L 5-aza can increase the rate of cardiomyocyte differentiation.Conclusion The combina