The efficiency of establishing pig pluripotent embryonic stem cell clones from blastocysts is still low. The transcription factor Nanog plays an important role in maintaining the pluripotency of mouse and human embryonic stem cells. Adequate activation of Nanog has been reported to increase the efficiency of establishing mouse embryonic stem cells from 3.5 day embryos. In mouse, Nanog starts to be strongly expressed as early as the morula stage, whereas in porcine NANOG starts to be strongly expressed by the late blastocyst stage. Therefore, here we investigated both the effect of expressing NANOG on porcine embryos early from the morula stage and the efficiency of porcine pluripotent embryonic stem cell clone formation. Compared with intact porcine embryos, NANOG overexpression induced a lower blastocyst rate, and did not show any advantages for embryo development and pluripotent embryonic stem cell line formation. These results indicated that, although NANOG is important pluripotent factor, NANOG overexpression is unnecessary for the initial formation of porcine pluripotent embryonic stem cell clones in vitro.