Here we report the productions of genetically modified cloned Massachusetts General Hospital miniature pig (MGH minipig) using freshly thawed donor cells equilibrated with roscovitine. Fibroblasts were isolated from the ear skin of a 10-day-old male MGH minipig. The donor cells were divided into two groups: cultured for 3 days (culture) and freshly thawed with 500 nM roscovitine. The viability of the donor cells was significantly higher at 0 h (94.6±3.5) compared with 1 h (81.7±5.7) after thawing (p=0.028). After 1 hr of equilibration time, the proportion of G0/G1 stage in roscovitine group was not different from 0 hr group, but not in culture medium group (p<0.01), respectively. Although the developmental characteristics were not different in both methods, the pregnancy and delivery rate in freshly thawed group were significantly higher than that of culture group (p<0.01), respectively. In total, 12 TG cloned MGH minipigs were delivered and the individual cloning efficiency was from 0 to 2.54%. Taken together, the use of freshly thawed donor cells equilibrated with roscovitine may be helpful method to increase the productivity of the genetically modified cloned MGH minipigs.