Background and Objectives Cellular cardiomyoplasty(CCM) is a promising treatment for ischemic cardiomyopathy.Up to now AZA (5-azacytidine) is the best known for MSC (mesenchymal stem cell) transdifferentiation to CMC (cardiomyocyte). But the best way of AZA treatment has not been established. In this study, we tried to find out the best protocol of AZA exposure to promote MSC differentiation to CMC. Method We used human MSC derived from umbilical cord blood. To detect MSC differentiation to CMC, we have done RT-PCR for CMC markers (?MHC, ANP, connexin 43, CTnI, GATA-4, MEF-2C). To establish optimal AZA exposure condition, we executed two phases of studies. In the 1st phase study, to obtain optimal concentration of AZA, we exposed MSC to AZA under 5 different schemes of concentration and time 1) 2.5 uM/L for 24 hrs exposure, 2) 2.5 uM/L for 72 hrs, 3) 2.5 uM/L for 120 hrs, 4) 10 uM/L for 24 hrs, 5) 10 uM/L for 72 hrs. And we did RT-PCR for CMC marker at 1, 2, 3, 4, 8 weeks in 5 conditions. After obtaining optimal AZA exposure condition, in the 2nd phase study we set up different pulse exposure conditions of AZA 2.5 uM/L as follows, 1) control (no exposure and harvest at Day 7), 2) harvest at Day 3 after AZA for 72hrs, 3) harvest at Day 7 after AZA for initial 72hrs, 4) harvest at Day 14 after 2 cyclic pulse exposure to AZA (one cycle=exposure for 72hrs and non-exposure for the subsequent 96 hrs), 5) harvest at Day 14 after continuous AZA exposure for 2 weeks. Results In the 1st phase of study, 2.5 uM/L of AZA exposure was the best condition for MSC differentiation to CMC-like cell. In RT-PCR, ANP and MEF-2C expressed 1 ?8 week, GATA 4 expressed 2-4 week, Connexin43 expressed 4-8 weeks, cTnI expressed 1-8 weeks. And in the 2nd phase of study, two repeated pulse exposure to 2.5uM/L AZA showed Connnexin-43, CTnI, GATA-4, MEF-2C expressed positively on 1, 2 weeks in RT-PCR. Especially early CMC maker (GATA-4) was expressed earlier than phase 1 condition. Conclusion For effective CCM, optimal MSC differentiation to CMC is essential. This pulse exposure method of AZA could be useful in application of MSC to myocardial regeneration in the future.