Abstract: Human cardiac α-actin promoter obtained from the human heart genome by polymerase chain reaction(PCR) was ligated into the same fragment of pEGFPN1（moved CMV promoter by AseI/Bgl II）.The constructed expression vector, named α-actin-pEGFP-N1 which contains neor gene and GFP gene as positive selection markers,was transferred into mice ES cells by Lipofectamine 2000 It’s found that DNA concentration should be 3.0-5.0μg/mL, transfer time was 2-3h, 200μg/mL G418 was used as perfect working concentration. Fluorescent observation and immunocytochemistry assay (PCNA and antibody of α-actin) demonstrated the transfected cells show high levels of α-actin and GFP expression, basically maitain its natural characteristic compared with wild mice ES cells. The results suggest that human cardiac αactin promoter may be useful in the study of mice ES cells specific differentiation into cardiomyocytes.